Team:Potsdam Bioware/Labjournal/September part 1

Contents 1 82th Labday 2011-09-01 1.1 Digest of pUP089 (vector) 1.2 miniprep of overnight cultures - mdnDE, mdnABC 1.3 Digest of miniprep - mdnDE, mdnABC 1.4 Testdigest of miniprep - mdnDE, mdnABC 1.5 Ligation of pUP089 and Lib-2 1.6 Transformation of library2 1.7 Picking clones for overnight cultures of pSB1C3 + mdnABC and DE, respectively 1.8 Digestion of NgoMIV_TEV-Protease_iGEM_BamHI, NgoMIV 14_3C-Protease_iGEM_BamHI, HindIII_iGEM_AraC_NgoMIV fragments and pUP189-pJC354-NheI-TEV-Xho_blaFL_GGH5 and pUP189-pJC354-NheI-143C-Xho_blaFL_GGH5 vector 1.9 Ligation of NgoMIV_iGEM_14_3C-Protease_iGEM_BamHI or NgoMIV_iGEM_TEV-Protease_iGEM_BamHI, HindIII_iGEM_AraC_NgoMIV into pJC354-NheI-143C-Xho_blaFL_GGH5 or pJC354-NheI-TEV-Xho_blaFL_GGH5 vector 1.10 plasmid pereperation and test digestion pPDV089 to control deletion of kanamycin gene, pARW089 containing only geneIII (no mdnA), pSB1C3 containing mdnA, geneIII or mdnA/geneIII 1.11 overnight culture of picked E. coli clones transformed with pARW089 containing only geneIII (no mdnA) 1.12 Production of phages containing pPDV089 2 83th Labday 2011-09-02 2.1 Miniprep of overnight cultures from ligation of pSB1C3 + mdnABC and mdnDE, respectively 2.2 test digest of pSB1C3 + mdnABC and DE, respectively 2.3 Colony PCR of plated cells from 01.09.2011 (see entry from 01.09.2011 for picked colonies) 2.4 over night cultures of pSB1C3+mdnABC clone 2 and P1 for screening team 2.5 plate transformation of pUP089A and B (XL1-blue) 2.6 PCR of mdnA with N-terminal myc tag (mcyN) 2.7 Transformation of XL1blue-cells with pPDV089 2.8 Send pSB1C3 vectors, containing geneIII, mdnA and geneIII/mdnA fusion gene, for sequencing 2.9 Plasmid preperation and test digestion of pARW089 containing only geneIII (no mdnA) 3 84th Labday 2011-09-03 3.1 PCR-clean up of mdnA with N-terminal myc tag (mcyN) (Jessica 2011-09-02) 3.2 Restriction enzyme digestion of mycN, mycC and pSB1C3 3.3 Ligation of mycC and mycN, respectively, and pSB1C3 3.4 Transformation of XL1 with mycC/mycN in pSB1C3 3.5 Oligo-Fillin for mdnA-Library 2 (repetition) 3.6 Gel purification of Oligo-Fillin for mdnA-Library 2 (repetition) 3.7 Restriction enzyme digestion of Oligo-Fillin for mdnA-Library 2 (repetition) and pUP089 3.8 Ligation of Lib2 (repetition) and pUP089 3.9 Transformation of XL1 with Lib2 in pUP089 3.10 Transformation of XL1 with K3 and A3 expression backbones 3.11 Picking clones for overnight cultures of pSB1C3 + DE clone 2 and clone 3 3.12 overnight culture of picked E. coli clones transformed with of XL1blue-cells with pPDV089 4 85th Labday 2011-09-04 4.1 Production of phages containing pPDV089 in XL1 blue cells 4.2 Miniprep of cells picked from plates on 02.09.2011 and inoculated into ON precultures 4.3 Miniprep of overnight cultures from ligation of pSB1C3+mdnDE clone 2 and clone3 4.4 Transformation of XL1 with mdnBC in pSB1C3 4.5 Control experiment: Transformation of XL1 with pARW089 and pARW071, respectively 4.6 Picking clones for overnight cultures of pSB1C3+mycC and pSB1C3+mycN 4.7 Picking clones for overnight cultures of pARW089 and pARW071 5 86th Labday 2011-09-05 5.1 Test digest of isolated plasmids (4.9.2011) from clones that were positive in colony PCR (2.09.2011) 5.2 competent cells - ER2738 5.3 check resistance of competent cells - E. coli ER2738 5.4 Miniprep of overnight cultures of pSB1C3+mycC and pSB1C3+mycN 5.5 Cultures of pARW089 and pARW071 for HPLC-Analysis 5.6 Oligo-Fillin for mdnA-Library 2 (repetition) 5.7 Control restriction enzyme digestion of pSB1C3_mdnA_mycC and pSB1C3_mdnA_mycN 5.8 PCR: mdnB, mdnABCDE 5.9 Different overnight cultures 5.10 Purification of phages containing pPDV089 5.11 Glycerol stocks of phages containing pPDV089 5.12 Concentration of phages containing pPDV089 measured by nanodrop 6 87th Labday 2011-09-06 6.1 Glycerol stocks of pSB1C3_mdnBC and pUP089 6.2 Miniprep of overnight cultures of pUP089 & psB1C3 + mdnBC 6.3 check plates - resistance of competent E. coli ER2738 cells 6.4 PCR mdnABCDE w/ Long PCR Enzyme mix from Fermentas 6.5 PCR purification of mdnB 6.6 Digest of purified PCR product mdnB and pSB1C3 6.7 PCR: Agarose Gel: mdnB digest verification; pSBIC3_B digest verification and PCR mdnABCDE with Long Enzyme Mix verification 6.8 PCR purification of PCR fragment mdnABCDE (T7, 89) and digested mdnB 6.9 PCR: Agarose Gel: pSBIC3_A, C, D, E 6.10 Gel Extraction of pSB1C3_E 6.11 Ligation of pSB1C3_E and mdnB 6.12 prepare samples for sequencing (created pARWIII) 6.13 Digestion of pSB1C3 6.14 digestion of vector pARWIII 6.15 Digestion of PCR products geneIII and mdnA for cloning into pSB1C3 6.16 Agarose gel electrophoresis for purification of digested PCR products and vectors 6.17 Ligation of geneIII and mdnA into pSB1C3 6.18 Re-ligation of NsiI-digested pARWIII 6.19 Transformation of created vectors in E. coli 6.20 PCR of TorA-bla with clevage site of 14_3C and TEV for BioBricks 6.21 PCR: mdnABCDE 6.22 Cultures of pARW071 and pARW089 for HPLC 6.23 Overnight cultures of expression backbones 7 88th Labday 2011-09-07 7.1 Digestion and Ligation of TorA-bla with cleavage site of 14_3C and TEV for BioBricks and TEV / 14_3C biobricks 7.2 Oligo-Fillin for mdnA-Library (repetition)2 7.3 Preparing samples mdnC, mdnD, mdnE and mdnABC for sequencing 7.4 Purification of Fillin mdnA-Lib2 7.5 digest of purified Fillin mdnA-Lib2 and pUP089 7.6 purification of digested pSB1C3 and pUP089 7.7 Ligation of Fillin mdnA-Lib2 and pUP089 7.8 Ligation of mdnABCDE in pSB1C3 7.9 Digest of purified PCR product mdnABCDE and pSB1C3 7.10 test digest of mdnBC 7.11 test pSB1A3_Ara_YFP and pSB1A3_Lac_YFP (expression backbones) 7.12 PCR: mdnABCDE 7.13 Overnight cultures of expression backbones and pUP089 7.14 Repeated test digest of mdnBC 7.15 Production of phages containing pPDV089 in ER2738 cells 7.16 SDS- PAGE for Western Blotting and coomassie staining of purified phages 7.17 Western Blot 7.18 Coomassie staining 7.19 overnight culture of picked E. coli clones transformed with pARWIII (pARW089 containing geneIII) to control deletion of kanamycin gene, pSB1C3 containing mdnA, geneIII or mdnA/geneIII 8 89th Labday 2011-09-08 8.1 Oligo-Fillin for mdnA-library 2 (repetition) and for Phage-display-mdnA-library (carrying AgeI restriction site) 8.2 Transformation of XL1 with different constructs 8.3 Purification of Fillin mdnA-Library_2 (carrying either AgeI or AatII restriction site) and PCR product of mdnABCDE from pARW071 8.4 Restriction enzyme digestion of mdnA focused library 2 carrying AatII restriction site and pUP089 8.5 Restriction enzyme digestion of mdnABCDE71 and pSB1C3 8.6 Restriction enzyme digestion of mdnA focused library 2 carrying AgeI restriction site and pARWIII 8.7 PCR of TorA-bla with clevage site of 14_3C and TEV for BioBricks 8.8 Test digest of pSB1C3+mdnDE 8.9 Overnight cultures of pSB1C3+myc 8.10 Sidedirected mutagenesis of TEV protease and 14_3C protease 8.11 Analytical gelelctrophoresis and PCR clean up of sidedirected mutated fragments T1, T2, P1, P2, P3 and P4 http://syntbio.net/iGEM/wiki2011/index.php/Labjournal_september#Sidedirected_mutagenesis_of_TEV_protease_and_14_3C_protease 8.12 Assembly PCR of mutated TEV protease and 14_3C protease fragments 8.13 Analytical gelelctrophoresis and PCR clean up of sidedirected mutated fragments TEV protease, P5 and P6 http://syntbio.net/iGEM/wiki2011/index.php/Labjournal_september#Assembly_PCR_of_mutated_TEV_protease_and_14_3C_protease_fragments 8.14 Preparing glycerol stocks of pUP089 8.15 Ligation of mdnA-lib2 and mdnA-Age 8.16 Purification of phages containing pPDV089 8.17 Glycerol stocks of phages containing pPDV089 8.18 Concentration of phages containing pPDV089 measured by nanodrop 8.19 plasmid pereperation of pARWIII to control deletion of kanamycin gene, pSB1C3 containing mdnA, geneIII or mdnA/geneIII 8.20 Antibody detection with blotted membranes 9 90th Labday 2011-09-09 9.1 Preparation of samples for HPLC analysis 9.2 Counting of colonies on plates (pUP089+lib2) 9.3 Glycerol stocks of pSB1C3+mdnDE K1, K2 and K5 9.4 Sequencing of pSB1C3+mdnD/ABC 9.5 Transformation of XL1 with different constructs 9.6 Survival-Test of one 14_3C clone( SG9-P10) and one Tev-clone (SG13-T6) 9.7 Assembly PCR of mutated 14_3C protease 9.8 Digestion of NgoMIV_TEV-Protease_iGEM_BamHI, NgoMIV_14_3C-Protease_iGEM_BamHI, HindIII_iGEM_AraC_NgoMIV fragments and pUP189-pJC354-NheI-TEV-Xho_blaFL_GGH5 and pUP189-pJC354-NheI-143C-Xho_blaFL_GGH5 vector 9.9 Ligation of NgoMIV_iGEM_14_3C-Protease_iGEM_BamHI or NgoMIV_iGEM_TEV-Protease_iGEM_BamHI, HindIII_iGEM_AraC_NgoMIV into pJC354-NheI-143C-Xho_blaFL_GGH5 or pJC354-NheI-TEV-Xho_blaFL_GGH5 vector 9.10 Miniprep from overnight cultures T I/II, 14_3C CS,PC, TEV-CS-1, P1 9.11 Sequencing of miniprep (09-09-2011) - T I/II, 14_3C CS,PC, TEV-CS-1, P1 9.12 Transformation of TEV and 14_3C 9.13 Test digest of pSB1C3+mycN/C 9.14 Miniprep of overnight cultures + pSB1C3+mycN/mycC 9.15 Sequencing of pSB1C3+mdnDE/mycN/mycC 9.16 Picking clones for overnight cultures of Lib 2 for confirmation 9.17 Prepare liquid culture for mdnA foc2 9.18 Picking clones for overnight cultures of pSB1C3+mdnABCDE with and without T7-promotor 9.19 Preparing overnight cultures of expression backbone-clones 9.20 PCR: mdnABCDE 9.21 test digestion to control if pSB1C3 is containing mdnA, geneIII or mdnA/geneIII 9.22 Send clones 2-6 (geneIII-mdnA in pSB) for sequencing 9.23 test digestion to control if kanamycin resistance gene is knocked out of pARWIII 10 91th Labday 2011-09-10 10.1 miniprep of Lib2 clones for confirmation 10.2 miniprep of mdnA foc2 10.3 miniprep of pSB1C3+mdnABCDE with and without T7 promotor 10.4 Counting of colonies on plates (pUP089+lib2) 10.5 New test digest of pSB1C3+mycN/C 10.6 Test Expression Backbones: pSB1K3_Ara/Lac_CFP 10.7 Glycerol stocks: Expression Backbones 10.8 Oligo-Fillin for mdnA-library 2 (repetition) and for Phage-display-mdnA-library (carrying AgeI restriction site) 10.9 Restriction enzyme digestion of ParWIII and fill-in-reaction of Libary 2 10.10 Restriction enzyme digestion of pSB1C3 for ligation with mycN/C 10.11 Restriction enzyme digestion of pSB1C3 for control 10.12 Picking clones for overnight cultures of pSB1C3+mdnABCDE 10.13 Preparing overnight cultures of expression backbone-clones 10.14 Transformation of mdnABCDE + pSB1C3 10.15 Colony PCR of plated TEV and 14_3C containing colonies 10.16 Digest of TEV, 14_3C, AraC and different Vectors (pJS354_ssTorA_XhoI_CS-TEV_NheI_blaFL, pJS354_ssTorA_XhoI_CS-TEV_NheI_blaFL and pSB1C3) 10.17 Gelextraction of digested fragements, ligation and transfomration of ligation products into E. coli XL1 blue 10.18 Ligation of digested fragments of TEV protease, 14_3C protease and different vectors 10.19 Transfromation of E. coli XL1 blue with ligation products 10.20 Plating of transformed E. coli XL1 blue

82th Labday 2011-09-01

Digest of pUP089 (vector)

Investigator: Niels, Katharina

Aim: generate vector for ligation and transformation to generate a libary

Materials/Methods:

• pub089 (A)

• • 5 µl DNA (~4000ng)

• • 0,5 µl Sfo I

• • 0,5 µl Aat II

• • 3 µl 10x Buffer 4 (Biolabs)

• • 21 µl water

• • • total volume: 30µl

Purification

• by NucleoSpin Extract II Kit, protocol for PCR purification

backseat driver asks: why do you use only 5 µl? what about gel purification?

Further tasks:

• dephosphorylation

• ligation

• transformation

miniprep of overnight cultures - mdnDE, mdnABC

Investigator: Niels, Jessica

Aim: Isolate DNA from over night cultures from 31.08.2011 (Katharina)

Materials/Methods:

1.miniprep:

• over night culture

• • mdnABC

• • • clone 3

• • • clone 4

• • • clone 5

• • • clone 6

• • • clone 7

• • mdnDE

• • • clone 1

• • • clone 2

• • • clone 3

• • • clone 4

• • • clone 5

• using Kit NucleoSpin® Plasmid (NoLid) (Macherey-Nagel)

• elution in 50µl Elution buffer

backseat driver: almost there, but a real plasmid name would be better.

2. Preparation of glycerol stocks:

• adding 300 µl glycerol to 700 µl culture
  

Result:

Further tasks:

• test-digest

Digest of miniprep - mdnDE, mdnABC

Investigator: Niels, Jessica

Aim: prove of isolated DNA from over night cultures from 2011-09-01 (Niels)

Materials/Methods:

• DNA (miniprep)

• SpeI

• EcoRI

• Buffer 4

• BSA

• Water

'1.Digest:

• DNA (µl) / Water (µl)

• • mdnABC

• • • clone 3 -

• • • clone 4 -

• • • clone 5 -

• • • clone 6 -

• • • clone 7 -

• • mdnDE

• • • clone 1 -

• • • clone 2 -

• • • clone 3 -

• • • clone 4 -

• • • clone 5 -

• 3µl Buffer 4 (Neblab)

• 0,3µl BSA

• 1 µl Spe I

• 1 µl EcoRI

• • incubate 2h at 37°C

Further tasks:

• analyze by gel

Testdigest of miniprep - mdnDE, mdnABC

Investigator: Niels, Katharina

Aim: prove of plasmid

Materials:

• DNA (miniprep)

• SpeI

• EcoRI

• Buffer 4

• BSA

• Water

Digest:
:

• mdnABC

• • 1µl DNA

• • 2 µl Buffer 2 (Neblab)

• • 0,5 µl HindIII

• • 0,5 µl AvaI

• • 16 µl Water

• • incubate 2h at 37°C

• mdnDE

• • 1µl DNA

• • 2 µl 10x Buffer 4 (Neblab)

• • 0,5µl HpaI

• • 16,5 µl Water

• • incubate 2h at 37°C

Further tasks:

Ligation of pUP089 and Lib-2

Investigators: Steffi, Niels, Katharina

Material

• purified pUP089 (vector)

• purified Lib-2 (insert)

backseat driver: do these poor pieces of DNA have no date or other referral?

Method

• 1 µl 10x Buffer

• 1 µl T4 Ligase

• 6 µl vector

• 2 µl insert

• 1h @ RT
  

Further tasks:

• transformation

Transformation of library2

Investigators:Katharina, Steffi

Aim: Transformation of Ligation

Materials:

• competent E. coli cells (XL1-Blue, 2011-08-29)

• ligation products: Lib2 + ligation control (2011-09-01, Nie, Kat, Ste)

Method:

• addition of 2 µl ligation reaction to cells (XL1-blue) in 1.5 ml Eppi,

• incubation 30 min on ice,

• heat shock 45 sec at 42°C,

• incubation 3 min on ice,

• addition of 750 µl LB medium,

• incubation at 37 °C shaking for 60 min,

• plating on LB medium with appropriate antibiotic (Kan)

• storage over night at 37°C

Further tasks:

• Picking clones for overnight culture

• Producing glycerol stocks

Output:

• 2 plates w/ Kan: mdn-lib2 + control

Picking clones for overnight cultures of pSB1C3 + mdnABC and DE, respectively

Investigators:Steffi, Katharina

Aim: check colonies for correct plasmid

Materials:

• agar plates from 2011-08-31, (fridge):

• • pSB1C3+mdnABC

• • pSB1C3+mdnDE

• LB medium

• chloramphenicol (25 mg/ml)

Protocol:

• 6 x: 5 ml LB medium + 5 µl chloramphenicol

• pick colony from plate (from each plate 3 colonies)

• transfer to LB medium

• incubate over night in 37°C shaker (200 rpm)

Output:

• over night cultures:

• • pSB1C3+mdnABC

• • • clone 1

• • • clone 2

• • • clone 3

• • pSB1C3+mdnDE

• • • clone 1

• • • clone 2

• • • clone 3

Further tasks:

• miniprep

• test digest

Digestion of NgoMIV_TEV-Protease_iGEM_BamHI, NgoMIV 14_3C-Protease_iGEM_BamHI, HindIII_iGEM_AraC_NgoMIV fragments and pUP189-pJC354-NheI-TEV-Xho_blaFL_GGH5 and pUP189-pJC354-NheI-143C-Xho_blaFL_GGH5 vector

For better understanding of described experiment see also: http://141.89.201.101/iGEM/wiki2011/images/c/c8/UP_SG_Klonierungsschema_Protease.pptx

Investigators:Paul, Sascha, Stefan, Sebastian

<td style="vertical-align: top; background-color: rgb(204, 204, 204);".</td></table> Materials:
4 samples NgoMIV_TEV-Protease_iGEM_BamHI:

• TEV 3: 71,7 ng/µl

• TEV 7: 65,3 ng/µl

• TEV 10: 58,6 ng/µl

• TEV 16: 62,0 ng/µl

2 samples NgoMIV_14_3C-Protease_iGEM_BamHI

• P1: 91,5 ng/µl

• P2: 82,5 ng/µl

2 samples HindIII_iGEM_AraC_NgoMIV

• A1: 103,7 ng/µl

• A2: 127,4 ng/µl

pUP189-pJC354-NheI-TEV-Xho_blaFL_GGH5 vector (contains TEV cleavage site): 470 ng/µl pUP189-pJC354-NheI-143C-Xho_blaFL_GGH5 vector (contains 14_3C cleavage site): 270,2 ng/µl

backseat driver: nice list, but dates would be really cool.

Digestion protocol:

1: NgoMIV_TEV-Protease_iGEM_BamHI (4x, each sample 1x):

• 10µl NgoMIV_iGEM_TEV-Protease_iGEM_BamHI

• 1µl NgoMIV

• 1µl BamHI-HF

• 5µl 10x buffer = NEB 4

• 0.5µl 100x BSA

• 32.5µl pure water

• =50µl

2: NgoMIV_14_3C-Protease_iGEM_BamHI (2x, each sample 1x):

• 10µl NgoMIV_14_3C-Protease_iGEM_BamHI

• 1µl NgoMIV

• 1µl BamHI-HF

• 5µl 10x buffer = NEB 4

• 0.5µl 100x BSA

• 32.5µl pure water

• =50µl

3: HindIII_iGEM_AraC_NgoMIV (2x, each sample 1x):

• 10µl HindIII_iGEM_AraC_NgoMIV

• 1µl HindIII

• 1µl NgoMIV

• 5µl 10x buffer = NEB4

• 0.5µl 100x BSA

• 32.5µl pure water

• =50µl

4: pUP189-pJC354-NheI-TEV-Xho_blaFL_GGH5:

• 3µl pUP189-pJC354-NheI-TEV-Xho_blaFL_GGH5

• 1µl HindIII

• 1µl BamHI-HF

• 5µl 10x buffer = NEB 4

• 0.5µl 100x BSA

• 39.5µl pure water

• =50µl

5: pUP189-pJC354-NheI-143C-Xho_blaFL_GGH5:

• 5µl pUP189-pJC354-NheI-143C-Xho_blaFL_GGH5

• 1µl HindIII

• 1µl BamHI-HF

• 5µl 10x buffer = NEB 4

• 0.5µl 100x BSA

• 37.5µl pure water

• =50µl

-->The reaction was allowed to proceed for 2h at 37°C!

Ligation of NgoMIV_iGEM_14_3C-Protease_iGEM_BamHI or NgoMIV_iGEM_TEV-Protease_iGEM_BamHI, HindIII_iGEM_AraC_NgoMIV into pJC354-NheI-143C-Xho_blaFL_GGH5 or pJC354-NheI-TEV-Xho_blaFL_GGH5 vector

For better understanding of described experiment see also: http://141.89.201.101/iGEM/wiki2011/images/c/c8/UP_SG_Klonierungsschema_Protease.pptx

Investigators: Paul, Sebastian, Sascha, Stefan

Aim:

• 1. Triple-ligation of NgoMIV_iGEM_14_3C-Protease_iGEM_BamHI (573bp), HindIII_iGEM_AraC_NgoMIV (1273bp) and pJC354-NheI-143C-Xho_blaFL_GGH5 vector (~4700bp) to create pUP_SG2_TorA_CS-14_3C_bla_AraC-14_3C
  

• 2.Triple-ligation of NgoMIV_iGEM_TEV_iGEM_BamHI (760bp), HindIII_iGEM_AraC_NgoMIV (1273bp) and pJC354-NheI-TEV-Xho_blaFL_GGH5 vector (~4700bp)to create pUP_SG1_TorA_CS-TEV_bla_AraC-TEV
  

Calculation of volumes to be used with: [http://www.gibthon.org/ligate.html ligation calculator] with 1:1 molar ratio

Materials:

14_3C: 2 reaction batches (we have two digested 14_3C-fractions)

1:

• 2,9 µL NgoMIV_iGEM_14_3C-Protease_iGEM_BamHI fragment (573bp, 5,4ng/µl)

• 2 µL AraC fragment (12.6 ng/µl)

• 3,1 µL pJC354-NheI-143C-Xho_blaFL_GGH5 vector (30.8 ng/µl)

• 1 µL T4 liagtion buffer (Fermentas)

• 1 µL T4 ligase (Fermentas)

• =10µl

2:

• 2,5 µL NgoMIV_iGEM_14_3C-Protease_iGEM_BamHI (573bp, 6,6 ng/µl) fragment

• 2,2 µL AraC fragment

• 3,3 µL pJC354-NheI-143C-Xho_blaFL_GGH5 vector (30.8 ng/µl)

• 1 µL T4 liagtion buffer (Fermentas)

• 1 µL T4 ligase (Fermentas)

• =10µl

• 1 controls: As 2 but with water instead of fragment

TEV: 4 reaction batches (we have three digested NgoMIV_iGEM_TEV-Protease_iGEM_BamHI fragment fractions)

1:

• 1.8 µL NgoMIV_iGEM_TEV-Protease_iGEM_BamHI fragment (760bp, 5,8 ng/µl)

• 1.4 µL AraC fragment (12,6 ng/µl)

• 4.9 µL pJC354-NheI-TEV-Xho_blaFL_GGH5 vector (12,9 ng/µl)

• 1 µL T4 liagtion buffer (Fermentas)

• 1 µL T4 ligase (Fermentas)

• =10µl

2:

• 2.4 µL NgoMIV_iGEM_TEV-Protease_iGEM_BamHI fragment (760bp, 3,7 ng/µl)

• 1.2 µL AraC fragment (12,6 ng/µl)

• 4.3 µL pJC354-NheI-TEV-Xho_blaFL_GGH5 vector (12,9 ng/µl)

• 1 µL T4 liagtion buffer (Fermentas)

• 1 µL T4 ligase (Fermentas)

• =10µl

3:

• 3.2 µL NgoMIV_iGEM_TEV-Protease_iGEM_BamHI fragment (760bp, 2.5 ng/µl)

• 1.1 µL AraC fragment (12,6 ng/µl)

• 3.8 µL pJC354-NheI-TEV-Xho_blaFL_GGH5 vector (12,9 ng/µl)

• 1 µL T4 liagtion buffer (Fermentas)

• 1 µL T4 ligase (Fermentas)

• =10µl

4:

• 2.4 µL NgoMIV_iGEM_TEV-Protease_iGEM_BamHI fragment (760bp, 3,9 ng/µl)

• 1.2 µL AraC fragment (12,6 ng/µl)

• 4.4 µL pJC354-NheI-TEV-Xho_blaFL_GGH5 vector (12,9 ng/µl)

• 1 µL T4 liagtion buffer (Fermentas)

• 1 µL T4 ligase (Fermentas)

• =10µl

backseat driver: are you doing the same thing four times? why do you fractionate your DNA?

• 1 control: As 4 but with water instead of fragment

Used method:

ligation at room temperatur for 1h

Further task: Transformation of XL1blue cells with ligation products

Edit:

Competent cells were transformed with the complete ligation batches and plated on Cm containing plates!

Results:

1: 14_3C containing colnes, 10 colonies picked

2: TEV containing clones, 10 colonies picked

Note: "Rest" means, that after plating 100µl on a plate remaining cells in a tube were centrifugated, resuspended and plated again on a new plate.

plasmid pereperation and test digestion pPDV089 to control deletion of kanamycin gene, pARW089 containing only geneIII (no mdnA), pSB1C3 containing mdnA, geneIII or mdnA/geneIII

Investigators: Sandrina, Laura

Aim: control deletion of kanamycin gene in created pPDV089, control ligation of geneIII into PARW089, control ligation of mdnA, geneIII and mdnA/geneIII into pSB1C3

Method/Materials:

plasmid preperation:

protocol 5.1 of the NucleoSpin Plasmid Kit

test digestion:

• 5 clones from pPDV089_2S14 (ampicillin):

• 4 µl (132-256 ng/µl) vector DNA

• 0.5 µl NsiI

• 0.5 µl SpHI

• 2 µl buffer 2

• 13 µl water

incubate for 1 h at 37°C

• 5 clones from pARW089 with geneIII (kanamycin):

test digestion could not be done, because cells did not grow over night

• 5 clones from pSB1C3 with mdnA (chloramphenicol), 5 clones from pSB1C3 with geneIII (chloramphenicol), 5 clones from pSB1C3 with mdnA/geneIII (chloramphenicol):

• 4 µl vector DNA (58- 120 ng/µl)

• 2 µl buffer 3

• 0,2 µl BSA

• 0.5 µl XbaI

• 0.5 µl PstI

12.8 µl water

incubate for 1 h at 37°C

Results:

Loading of gels

lane	Sample	Volume in µl	Expected insert size in bp
M	marker, DNA ladder mix Fermentas
1	geneIII in pSB1C3, clone 1	10	ca. 500
2	geneIII in pSB1C3, clone 2	10	ca. 500
3	geneIII in pSB1C3, clone 3	10	ca. 500
4	geneIII in pSB1C3, clone 4	10	ca. 500
5	geneIII in pSB1C3, clone 5	10	ca. 500
6	free
7	mdnA in pSB1C3, clone 1	12	ca. 180
8	mdnA in pSB1C3, clone 2	12	ca. 180
9	mdnA in pSB1C3, clone 3	12	ca. 180
10	mdnA in pSB1C3, clone 4	12	ca. 180
11	mdnA in pSB1C3, clone 5	12	ca. 180
12	pPDV089, clone 1	12	ca. 1400
13	pPDV089, clone 2	12	ca. 1400
14	pPDV089, clone 3	12	ca. 1400
15	pPDV089, clone 4	12	ca. 1400
16	pPDV089, clone 5	12	ca. 1400
17	free
18	mdnA-geneIII fusion gene in pSB1C3, clone 1	12	ca. 700
19	mdnA-geneIII fusion gene in pSB1C3, clone 2	12	ca. 700
20	mdnA-geneIII fusion gene in pSB1C3, clone 3	12	ca. 700
21	mdnA-geneIII fusion gene in pSB1C3, clone 4	12	ca. 700
22	mdnA-geneIII fusion gene in pSB1C3, clone 5	12	ca. 700

Further tasks:

• sequncing of geneIII in pSB1C3, clone 3, mdnA-geneIII fusion gene in pSB1C3, clone 4 and mdnA in pSB1C3, clone 4

• phage display with pPDV089

• repeat ligation of geneIII in pARW089

overnight culture of picked E. coli clones transformed with pARW089 containing only geneIII (no mdnA)

Investigators: Sandrina, Laura

Aim: control ligation of geneIII into PARW089

Method/Materials:

• 4 clones from pARW089 with geneIII (kanamycin)

• 5 ml LB medium per clone

• storage over night at 37°C and 750 rpm
  

Further tasks:

• test digestion
  

Production of phages containing pPDV089

Investigators: Sandrina, Laura

Aim: control if geneIII-mdnA will be expressed on the phage

Method/Materials:

• first: amplification of cells containing pPDV089

• 50 ml DYT medium will be inoculated with the cells, so that OD600 = 0.1

• antibiotics tetracyclin and ampicillin should be geiven to the medium

• cells should be incubated 37 °C till OD600 = 0.3-0.5 is reached

• upcomming problem: cells died

Further tasks:

• repeat this procedure
  

83th Labday 2011-09-02

Miniprep of overnight cultures from ligation of pSB1C3 + mdnABC and mdnDE, respectively

Investigators: Vanessa, Katharina

Time: 2011-09-02

Aim: DNA for sequencing and confirmation of insert

Materials:

• 6 overnight cultures

• NucleoSpin® Plasmid (NoLid) (Macherey-Nagel)

• • Protocol for high-copy plasmids

• • elution with 50 µl H₂O

• measuring concentration with NanoDrop:

Sample	concentration in ng/µl
mdnABC clone 1	745.7
mdnABC clone 2	1121.8
mdnABC clone 3	974.6
mdnDE clone 1	873.2
mdnDE clone 2	665.5
mdnDE clone 3	665.7

test digest of pSB1C3 + mdnABC and DE, respectively

Time: 2011-09-02

Investigators: Steffi, Katharina

Aim: prove of Insert (mdnABC or mdnDE)

Materials:

Digestion protocol for pSB1C3+mdnABC

• 1 µl DNA

• 0.3 µl BSA

• 1 µl HincII

• 3 µl 10x buffer = NEB 3

• 24.7 µl pure water

• 37°C for 1h

Digestion protocol pSB1C3+mdnDE

• 1 µl DNA

• 1 µl HpaI

• 3 µl 10x buffer = NEB 4

• 25 µl pure water

• 37°C for 1h

Production of one 1 %

• 1 % gel: 0.5 g agarose in 50 ml 1x TAE buffer

• Adding 2 µl gel red to each gel

Loading gels and running

Loading of gels

lane	Sample	Volume in µl	Expected size in bp
M	marker
1	mndABC clone 1	12	486, 1482, 2884
2	mdnABC clone 2	12	486, 1482, 2884
3	mdnABC clone 3	12	486, 1482, 2884
4	mdnDE clone 1	12	1329, 3825
5	mdnDE clone 2	12	1329, 3825
6	mdnDE clone 3	12	1329, 3825

Results:

Conclusions:

• test digest seems to be correct for:

• • lane 2, 5 and 6

Further task:

• send positive plasmids for sequencing

Colony PCR of plated cells from 01.09.2011 (see entry from 01.09.2011 for picked colonies)

Investigators: Sascha, Paul, Sebastian

Aim:

Proove whether triple ligation of TEV and 14_3C into backbone was succesful.

Used Primer:

Tev:

f_AraC_HindIII_iGEM, r_TEV_iGEM_BamHI

14_3C:

f_AraC_HindIII_iGEM, r_14_3C_iGem:BamHI

Method:

• 2,5µl of each primer (10µM) = 5µl

• 5µl 10y polymerase buffer

• 2µl 25mM MgCl2

• 1µl dNTP mix

• 0,5µl Taq-Polymerase (GenAxxon)

• 36,5 µl H2O

=50µl

Colonies were picked with a 20µl tip, dipped into PCR batch, and then plunged into 1ml LB to grew cells from picked colony (from these 1ml batches overnight cultures will be inoculated in case of positive clones).

• 10 colonies were picked for TEV and 10 colonies for 14_3C protease (see entry from 01.09.2011) = 20 PCR batches.

PCR Program:

Initial denat = 3min 94°C

25x

denat: 2min 10sec 94°C

anneal: 2min 10sec 70°C

extend: 2min 10sec 72°C

final extend: 10min 72°C

• The PCR products were resolved on a 1% analytical agarose gel

Expected fragments:

Tev+AraC ~ 2033 bp

14_3C+AraC ~ 1846 bp

Results:

400px

• Based on the results from the agarose gel overnight precultures from the 1 ml batches were prepared.

Following clones were used:

Tev:

T2, T4, T5, T6, T7, T8, T9, T10

14_3C:

P1, P3, P4, P6; P8, P9, P10

over night cultures of pSB1C3+mdnABC clone 2 and P1 for screening team

time: 2011-9-2, 18:00

Investigators: Nadine, Jessica

Aim: preparation of gycerol stock from pSB1C3+mdnABC transformed cells (cultures were placed instable in the incubator)

Materials:

• over night culture from Katharina (2011-9-1)

• • pSB1C3+mdnABC clone 2

• over night culture from Paul (2011-9-2):

• • P1

• LB medium

• chloramphenicol (25 mg/ml)

Protocol:

• 2 x: 10 ml LB medium + 10 µl chloramphenicol

• use left cell suspension for inoculation

• transfer to LB medium

• incubate over night in 37°C shaker (200 rpm)

Output:

• over night cultures:

• • pSB1C3+mdnABC

• • • clone 3

• • P1 for screening team

Further tasks:

• mdnABC: glycerol stocks

plate transformation of pUP089A and B (XL1-blue)

time: 2011-9-2, 17:00

Investigators: Nadine, Jessica

Aim: get new pUP089 vector that is not contaminated, check new plasmid for antibiotic resistance

Materials:

• Trafo from Jessica (2011-9-2)

• • pUP089 A

• • pUP089 B

• Agar plates w/:

• • 100 µg/ml Cm (2x)

• • 100 µg/ml Amp (1x)

• • 100 µg/ml Kan (2x)

Protocol:

• centrifuge incubated cells

• resuspend in 16 µl LB

• plate 50 µl

• incubate over night in 37°C

Output:

• Agar plates:

• • Cm pUP089 A

• • Cm pUP089 B

• • Kan pUP089 A

• • Kan pUP089 B

• • Amp pUP089 A

Further tasks:

• check plates

• over night cultures

PCR of mdnA with N-terminal myc tag (mcyN)

Investigators: Jessica

Time: 2011-09-02, 14:00

Material:

• pARW089 (~8 ng/µl)

• dNTPs

• primer 23, 24

• HF Phusion Buffer 5x

• Phusion Polymerase

Method:

• 1 µl pARW089

• 1 µl dNTPs (10 mM each)

• 2.5 µl forward primer (10µM)

• 2.5 µl reverse primer (10µM)

• 10 µl HF Phusion Buffer 5x

• 0.5 µl Phusion Polymerase

• 32.5 µl water

• total 50 µl

• edited programm IGBIO2

Step	Temperature	Time
Hot Start	98°C	Hold
Initial denaturation	98°C	30 sec
Denaturation	98°C	10 s	10x
Annealing	58°C	20 s
Extension	72°C	20 s
Denaturation	98°C	10 s	20x
Annealing	72°C	20 s
Extension	72°C	20 s
Final extension	72°C	10 min
	4°C	Hold

Result:

• PCR product mycN (stored in fridge, violett reck)

NOTE: PCR mycC done in july is stored at -20°C in blue PCR box (gel purified)

Further Tasks:

• gel electrophoresis

• PCR clean up

• restriction enzyme digestion

• ligation

• transformation

Transformation of XL1blue-cells with pPDV089

Investigators: Sandrina

Aim: control if geneIII-mdnA will be expressed on the phage

Method/Materials:

• addition of 0,3 µl ligated vector pPDV089 from clone 2S14 (after plasmid preperation) to competent XL1-blue cells
  

• incubation 25 min on ice,
  

• heat shock 45 sec at 42°C,
  

• incubation 2 min on ice,
  

• addition of 750 µl LB medium,
  

• incubation 60 min at 37 °C and 750 rpm
  

• plating on agar plates containing 100 µg/ml tetracyclin and 100 µg/ml ampicillin
  

• storage over night at 37°C

Further tasks:

• over night culture, amplification of cells for phage display, test digestion
  

Send pSB1C3 vectors, containing geneIII, mdnA and geneIII/mdnA fusion gene, for sequencing

Investigators: Sandrina

Aim: control if mdnA, geneIII and mdnA/geneIII fusion gene were fully ligated in pSB1C3 to generate biobricks

Method/Materials:

• sending clones geneIII in pSB1C3, clone 3, mdnA-geneIII fusion gene in pSB1C3, clone 4 and mdnA in pSB1C3, clone 4 to GATC

• 20 µl, 50-100 ng/µl

• primer:

Further tasks:

• sequence alignment
  

Plasmid preperation and test digestion of pARW089 containing only geneIII (no mdnA)

Investigators: Sandrina

Aim: control if ligation of geneIII in pARW089 worked

Method/Materials:

plasmid preperation:

• protocol 5.1 of the NucleoSpin Plasmid Kit

• test digestion:

• 4 clones from pARW089 with geneIII:

• 4 µl (132-256 ng/µl) vector DNA

• 0.5 µl SfoI

• 0.5 µl AatII

• 2 µl buffer 4

• 13 µl water

• incubate for 1 h at 37°C

• glycerol stocks of clone 1, 2 and 4 (stored at -80°C)

Results:

Loading of gels

lane	Sample	Volume in µl	Expected size in bp
M	marker, DNA ladder mix Fermentas
1	geneIII in pARW089, clone 1	10	ca. 10000 and 550
2	geneIII in pARW089, clone 2	10	ca. 10000 and 550
3	geneIII in pARW089, clone 3	10	ca. 10000 and 550
4	geneIII in pARW089, clone 4	10	ca. 10000 and 550

Further tasks:

• sequence vector
  

84th Labday 2011-09-03

PCR-clean up of mdnA with N-terminal myc tag (mcyN) (Jessica 2011-09-02)

Investigators: Nadine, Katharina

Time: 2011-09-03, 9:00

Material:

• PCR product of mycN

• NucleoSpin Extract II Kit from Macherey-Nagel

• elution in 50 µl

Result:

• concentration of product:

• • 40.0 ng/µl

Restriction enzyme digestion of mycN, mycC and pSB1C3

Investigators: Nadine, Katharina

Time: 2011-09-03, 10:00

Material:

• purified PCR products

• • mycC (4.8 ng/µl (2011-07-05 Jessica))

• • mycN (40.0 ng/µl)

• NEB Buffer 4

• XbaI

• PstI

• BSA

• • digestion of mycN and mycC

• • • 30 µl DNA

• • • 5 µl Buffer 4

• • • 1.5 µl XbaI

• • • 2.0 µl PstI

• • • 0.5 µl BSA

• • • 12 µl water

• • digestion of pSB1C3

• • • 6 µl DNA (235.0 ng/µl (2011-06-22))

• • • 3 µl Buffer 4

• • • 1.5 µl XbaI

• • • 2.0 µl PstI

• • • 0.5 µl BSA

• • • 11.5 µl water

• concentration after digestion

• • mycC: 4.2 ng/µl

• • mycN: 15.2 ng/µl

• • pSB1C3: 16.4 ng/µl

Ligation of mycC and mycN, respectively, and pSB1C3

Time: 2011-09-03, 17:30

Investigators: Nadine, Katharina

Materials

• T4 DNA Ligase Buffer (Fermentas)

• T4 DNA Ligase

• purified samples of Lib2 and pUP089

Method mycC

• 1µl T4 DNA Ligase Buffer

• 1µl T4 DNA Ligase

• 5µl pSB1C3

• 3µl purified mycC

• also preparing ligation control (3µl water instead of mycC)

• incubation for 1h at roomtemperature

Method mycN

• 1µl T4 DNA Ligase Buffer

• 1µl T4 DNA Ligase

• 7µl pSB1C3

• 1µl purified mycN

• incubation for 1h at roomtemperature

Transformation of XL1 with mycC/mycN in pSB1C3

Time: 2011-09-03, 19:00

Investigators: Nadine, Katharina

Materials:

• XL1-Blue

• ligation products: pSB1C3+mycC and pSB1C3+mycN

Method:

• addition of 2 µl ligation reaction to cells (XL1-blue) in 1.5 ml Eppi,

• incubation 25 min on ice,

• heat shock 90 sec at 42°C,

• incubation 5 min on ice,

• addition of 750 µl LB medium,

• incubation at 37 °C shaking for 60 min,

• plating on LB medium with appropriate antibiotic (Kan)

• storage over night at 37°C

Further tasks:

• Picking clones for overnight culture

• Producing glycerol stocks

Output:

• 2 plates with Cm: mycC, mycN

• 1 plate with Cm: control myc

Oligo-Fillin for mdnA-Library 2 (repetition)

Time: 2011-09-03, 11:00

Investigators: Nadine, Katharina

Materials

• Primer, 25 µM :

• • # 74: o_foc_library_2

• Klenow-Buffer 10X

• Klenow Fragment

• dNTPs

• water

Protocol:

• Reaction mix 1

• • 1 µl fw-Oligonucleotide (#76)

• • 1 µl rev-Oligonucleotide (#74)

• • 2 µl dNTPs (10 mM)

• • 2 µl Klenow-Buffer

• • 0.5 µl Klenow Fragment

• • 14 µl water

• • total volume: 20.5 µl

• Reaction mix 2

• • 2 µl fw-Oligonucleotide (#76)

• • 2 µl rev-Oligonucleotide (#74)

• • 2 µl dNTPs (10 mM)

• • 2 µl Klenow-Buffer

• • 0.5 µl Klenow Fragment

• • 12 µl water

• • total volume: 20.5 µl

• reaction mix 3

• • 3 µl fw-Oligonucleotide (#76)

• • 3 µl rev-Oligonucleotide (#74)

• • 2 µl dNTPs (10 mM)

• • 2 µl Klenow-Buffer

• • 0.5 µl Klenow Fragment

• • 10 µl water

• • total volume: 20.5 µl

2. PCR program

• name: Fillin

• 3 min 94 °C

• 0.3°C per s (94°C-37°C)

• addition of 0.5 µl Klenow Fragment

• press enter

• 1hr 37°C

Agarose gel:

1 %: 0.5 g in 50 ml TAE

Results:

• Output:

• • • mdnA-Lib2 1µl

• • • mdnA-Lib2 2µl

• • • mdnA-Lib2 3µl

Further tasks:

• Gel purification

Gel purification of Oligo-Fillin for mdnA-Library 2 (repetition)

Time: 2011-09-03, 13:00

Investigators: Nadine, Katharina

Materials

• Macherey Nagel - NucleoSpin Extract II, protocol for DNA extraction from agarose gels

• Eluation with 50µl NE-Buffer
  

NanoDrop: Concentrations

• Lib2 (1 µl) purified: 13.8 ng/µl

• Lib2 (2 µl) purified: 14.9 ng/µl

• Lib2 (3 µl) purified: 9.0 ng/µl

Restriction enzyme digestion of Oligo-Fillin for mdnA-Library 2 (repetition) and pUP089

Time: 2011-09-03, 13:00

Investigators: Nadine, Katharina

Materials

• purified Lib2 samples

• pUP089 (2011-06-22)

• AatII

• NEB Buffer 4

Method

Digestion protocol for Lib2

• 50 µl purified Lib2 sample

• 2.0 µl AatII

• 6 µl Buffer 4

• 2.0 µl water

• 37°C for 1 h

Digestion protocol for pUP089:

• pUP089

• NEB Buffer 4

• SfoI

• AatII

• • digestion protocol

• • • 4 µl DNA

• • • 3µl Buffer 4

• • • 1.5 µl AatII

• • • 1.5 SfoI

• • • 20 µl water

• • • incubation @ 37°C for 4 h

• • control digestion 1

• • • 2 µl DNA

• • • 3 µl Buffer 4

• • • 1.5 µl AatII

• • • 23.5 µl water

• • • incubation @ 37°C for 4 h

• • control digestion 2

• • • 2 µl DNA

• • • 3 µl Buffer 4

• • • 1.5 µl SfoI

• • • 23.5 µl water

• • • incubation @ 37°C for 1h

Loading of gels

lane	Sample
M	marker, DNA ladder mix Fermentas
1	Lib2 (1µl) digested
2	Lib2 (2µl) digested
3	Lib2 (3µl) digested
4	pUP SfoI/AatII
5	pUP AatII (control)
6	pUP SfoI (control)

• fragments were excised and purified using Macherey-Nagel Nucleo SpinII Extract Kit

• concentrations after purification

• • Lib2 (1µl): 6.8 ng/µl

• • Lib2 (2µl): 4.7 ng/µl

• • Lib2 (3µl): 4.9 ng/µl

• • pUP089: 14.9 ng/µl

Ligation of Lib2 (repetition) and pUP089

Time: 2011-09-03, 17:30

Investigators: Nadine, Katharina

Materials

• T4 DNA Ligase Buffer (Fermentas)

• T4 DNA Ligase

• purified samples of Lib2 and pUP089

Method

• 1µl T4 DNA Ligase Buffer

• 1µl T4 DNA Ligase

• 7µl pUP089

• 1µl Lib2 (1µl)

• also preparing ligation control (1µl water instead of Lib2)

• incubation for 1h at roomtemperature

Transformation of XL1 with Lib2 in pUP089

Time: 2011-09-03, 19:00

Investigators: Nadine, Katharina

Materials:

• XL1-Blue

• ligation products: pUP089+Lib2

Method:

• addition of 2 µl ligation reaction to cells (XL1-blue) in 1.5 ml Eppi,

• incubation 25 min on ice,

• heat shock 90 sec at 42°C,

• incubation 5 min on ice,

• addition of 750 µl LB medium,

• incubation at 37 °C shaking for 60 min,

• plating on LB medium with appropriate antibiotic (Kan)

• storage over night at 37°C

Further tasks:

• Picking clones for overnight culture

• Producing glycerol stocks

Output:

• 10 plates with Kan: Lib2

• 1 plate with Kan: control Lib2

Transformation of XL1 with K3 and A3 expression backbones

Time: 2011-09-03, 19:00

Investigators: Nadine, Katharina

Materials:

• XL1-Blue

• DNA of:

• • pSB1A3_YFP_Ara clone A

• • pSB1A3_YFP_Lac clone B

• • pSB1K3_CFP_Ara clone A

• • pSB1K3_CFP_Lac clone B

• • pSB1K3_YFP_Lac clone C

Method:

• addition of 2 µl DNA to cells (XL1-blue) in 1.5 ml Eppi,

• incubation 25 min on ice,

• heat shock 45 sec at 42°C,

• incubation 2 min on ice,

• addition of 750 µl LB medium,

• incubation at 37 °C shaking for 60 min,

• plating on LB medium with appropriate antibiotic (Kan/Amp)

• storage over night at 37°C

Further tasks:

• Picking clones for overnight culture

• Producing glycerol stocks

Picking clones for overnight cultures of pSB1C3 + DE clone 2 and clone 3

Investigators:Sebastian, Katharina

Time: 2011-09-04, 16:00

Materials:

• liquid cultures of mdnDE clone 2 and mdnDE clone 3

• LB medium

• chloramphenicol (25 mg/ml)

Method:

• inoculate 1 ml of liquid culture to 4 ml LB medium+ Cm

• incubate over night in 37°C shaker (200 rpm)

Further tasks:

• miniprep

• sending for sequencing

overnight culture of picked E. coli clones transformed with of XL1blue-cells with pPDV089

Investigators: Sandrina

Aim: control if geneIII-mdnA will be expressed on the phage

Method/Materials:

• 3 clones from pPDV089, clone 2S14, (ampicillin and tetracyclin)

• 5 ml LB medium per clone

• storage over night at 37°C and 750 rpm
  

Further tasks:

• amplification for phage display and test digestion
  

85th Labday 2011-09-04

Production of phages containing pPDV089 in XL1 blue cells

Investigators: Sandrina, Katharina, Nadine

Aim: control if geneIII-mdnA will be expressed on the phage

Method/Materials:

• first step: amplification of cells containing pPDV089( clone: 2S14):

• 50 ml DYT medium will be inoculated with the cells, so that OD600 = 0.1

• add antibiotics tetracyclin and ampicillin to the medium

• cells should be incubated 37 °C till OD600 = 0.3-0.5 (here: 0.332) is reached

• second step: infection with helper phages

• add helper phages 10^11 phages/50 ml (...)

• incubate for 10 min at 37°C (without shaking!)

• add 0,5 mM IPTG

• incubate 50 min at 28°C and rpm

• add 70 µg/ml kanamycin and incubate for 5 h at 28°C (...)

• third step: phage purification

• centrifuge cell culture at 5000 x g/ 15 min

• fill supernatant in a new 50 ml falcon and centrifuge again (5000 g/15 min)

• 40 ml of the supernatant with 8 ml PEG-NaCl (20% (w/v) PEG-8000, 2,5 M NaCl)

• incubate over night at 4°C

Further tasks:

• go on with phage purification
  

Miniprep of cells picked from plates on 02.09.2011 and inoculated into ON precultures

Investigators: Sandrina, Katharina

Time: 2011-09-04, 11:00

1. Miniprep:

• 15 overnight cultures (pUP SG3- pUP SG17)

• NucleoSpin® Plasmid (NoLid) (Macherey-Nagel)

• • Protocol for high-copy plasmids

• • elution with 50 µl H₂O

• measuring concentration with NanoDrop:

Sample	concentration in ng/µl
pUP SG3	1410.2
pUP SG4	1146.2
pUP SG5	1289.7
pUP SG6	940.1
pUP SG7	1418.5
pUP SG8	1244.4
pUP SG9	1287.6
pUP SG10	1188.7
pUP SG11	1276.1
pUP SG12	1193.9
pUP SG13	1255.1
pUP SG14	1474.7
pUP SG15	1143.5
pUP SG16	1268.0
pUP SG17	1218.7

Miniprep of overnight cultures from ligation of pSB1C3+mdnDE clone 2 and clone3

Investigators: Sandrina, Katharina

Time: 2011-09-04, 11:00

Material:

• 2 overnight cultures (mdnDE clone 2 and mdnDE clone 3)

• NucleoSpin® Plasmid (NoLid) (Macherey-Nagel)

• • Protocol for high-copy plasmids

• • elution with 50 µl H₂O

Results:

• measuring concentration with NanoDrop:

Sample	concentration in ng/µl
mdnDE clone 2	601.9
mdnDE clone 3	571.7

Further Tasks:

• sending for sequencing

Transformation of XL1 with mdnBC in pSB1C3

Time: 2011-09-04, 14:00

Investigators: Nadine, Katharina

Materials:

• XL1-Blue

• ligation products: pSB1C3+mdnBC (Niels 2011-08-24)

Method:

• addition of 2 µl ligation reaction to cells (XL1-blue) in 1.5 ml Eppi,

• incubation 25 min on ice,

• heat shock 45 sec at 42°C,

• incubation 2 min on ice,

• addition of 750 µl LB medium,

• incubation at 37 °C shaking for 60 min,

• plating on LB medium with appropriate antibiotic (Cm)

• storage over night at 37°C

• no ligation control available
  

Further tasks:

• Picking clones for overnight culture

• Producing glycerol stocks

Control experiment: Transformation of XL1 with pARW089 and pARW071, respectively

Time: 2011-09-04, 14:00

Investigators: Nadine, Katharina

Materials:

• XL1-Blue

• DNA of pARW089 and pARW071 (2011-05-24)

Method:

• addition of 2 µl ligation reaction to cells (XL1-blue) in 1.5 ml Eppi,

• incubation 25 min on ice,

• heat shock 45 sec at 42°C,

• incubation 2 min on ice,

• addition of 750 µl LB medium,

• incubation at 37 °C shaking for 60 min,

• plating on LB medium with appropriate antibiotic (Cm)

• storage over night at 37°C

• no ligation control available
  

Picking clones for overnight cultures of pSB1C3+mycC and pSB1C3+mycN

Investigators:Katharina

Time: 2011-09-04, 16:00

Materials:

• agar plates of pSB1C3+mycC and pSB1C3+mycN

• LB medium

• chloramphenicol (25 mg/ml)

Method:

• picking 5 clones for each construct

• incubate over night in 37°C shaker (200 rpm)

Output:

• 5 liquid cultures of mycC (1-5)

• 5 liquid cultures of mycN (1-5)

Further tasks:

• miniprep

• sending for sequencing

Picking clones for overnight cultures of pARW089 and pARW071

Investigators:Katharina

Time: 2011-09-04, 16:00

Materials:

• Glycerolstocks of pARW089 and pARW071

• LB medium

• Kan

Method:

• preparing one liquid culture for each construct

• incubate over night in 37°C shaker (200 rpm)

Further tasks:

• preparation of 400ml culture for HPLC analysis on 2011-09-05
  

86th Labday 2011-09-05

Test digest of isolated plasmids (4.9.2011) from clones that were positive in colony PCR (2.09.2011)

Investigators: Paul, Stefan, Sebastian

Aim: Prove whether plasmids contain all expected inserts.

Method: Digestion of plasmids with HincII. Produces 3 fragments from both, the 14_3C and the TEV containing plasmids.

• Expected fragments:

• • Tev: 550 bp, 2700 bp, 3400 bp

• • 14_3C: 550 bp, 2300 bp, 3600 bp

• 5µl 10x buffer 4 (NEB)

• 0,5µl 100x BSA

• 1µl Sample

• 0,5µl HincII Enzyme (NEB)

• 43µl water

• • 15 reaction batches, 7 for 14_3C, 8 for TEV:

following colnes were used:

2 pUP_SG3_ssTorA_CS-14_3C_blaFL_AraC-14_3C1

3 pUP_SG4_ssTorA_CS-14_3C_blaFL_AraC-14_3C3

4 pUP_SG5_ssTorA_CS-14_3C_blaFL_AraC-14_3C4

5 pUP_SG6_ssTorA_CS-14_3C_blaFL_AraC-14_3C6

6 pUP_SG7_ssTorA_CS-14_3C_blaFL_AraC-14_3C8

7 pUP_SG8_ssTorA_CS-14_3C_blaFL_AraC-14_3C9

8 pUP_SG9_ssTorA_CS-14_3C_blaFL_AraC-14_3C10

11 pUP_SG10_ssTorA_CS-TEV_blaFL_AraC-TEV2

12 pUP_SG11_ssTorA_CS-TEV_blaFL_AraC-TEV4

13 pUP_SG12_ssTorA_CS-TEV_blaFL_AraC-TEV5

14 pUP_SG13_ssTorA_CS-TEV_blaFL_AraC-TEV6

15 pUP_SG14_ssTorA_CS-TEV_blaFL_AraC-TEV7

16 pUP_SG15_ssTorA_CS-TEV_blaFL_AraC-TEV8

17 pUP_SG16_ssTorA_CS-TEV_blaFL_AraC-TEV9

18 pUP_SG17_ssTorA_CS-TEV_blaFL_AraC-TEV10

Results:

400px

competent cells - ER2738

Investigator: Katharina, Niels, Sandrina, Steffi

Aim: produce competent cells

Materials/Methods:

TFB I	1000ml	200ml
100mM Rubidium Chloride	12.1	2.42g
30mM Potassium Acetate	2.944	0.59g
10mM Calcium Chloride	1.47	0.29g
15% w/v Glycerol (87%)	150	34.5g

Adjust pH to 5.8 with acetic acid

Filter sterilize the solution

TFB II	500ml	100ml
50mM Rubidium Chloride	0.6	0.121g
10mM MOPS	1.05	0.210g
75mM Calcium Chloride	5.51	1.100g
15% w/v Glycerol (87%)	75	17.24g

Adjust pH to 7.0 with KOH

Filter sterilize the solution

Work always sterile and cold and speedy!

• All volumes deal with the common cellline!

• Prepare 80 Eppis (1,5?l)

• get liquid nitrogen

• prepare 5 ml LB-Medium with the specific antibiotic (for ER2738: Tet), inoculate and incubate over night

• prepare 200 ml LB-Medium with the specific antibiotic, inoculate with 2 ml of the over-night-culture

• grow while shaking at 37°C, 190 rpm to an OD600 at 0,4-0,6

• keep cell suspension in sterile falcons (50 ml) 10 min on ice, then centrifuge for 5 min, 4°C, 4000 rpm

• discard supernatant, carefully resuspend on ice with 40 ml icecold TFB I and keep 10 min on ice

• centrifuge for 5 min, 4°C, 4000 rpm

• discard supernatant, carefully resuspend pellet in 8 ml TFB II

• aliquot in Eppis: 50?l per tube and store immediately at liquid nitrogen and afterwards at -80 °C

Results:

• 80 tubes ER2738 for transformation(50 µl competent cells at -80°C)

Further tasks:
check by transformation and check the resistance on agar plates with different antibiotics

check resistance of competent cells - E. coli ER2738

Investigator: Niels, Katharina, Sandrina, Steffi

Aim: check resistance of competent ER2738-cells on agar plates with different antibiotics

Materials/Methods:

• • competent ER2738-cells from 2011-09-05 (Niels, Katharina, Sandrina, Steffi)

• • LB-plates with Tetracycline, Chloramphenicol, Kanamycine, Ampicillin, without antibiotics

• • plating 50 µl on agar plates

• • incubate at 37°C over night

Further tasks:
control agar plates

Miniprep of overnight cultures of pSB1C3+mycC and pSB1C3+mycN

Investigators:Nadine, Nicole, Katharina

Time: 2011-09-04, 9:00

Materials:

• liquid culture of pSB1C3+mycC and pSB1C3+mycN

• NucleoSpin® Plasmid (NoLid) (Macherey-Nagel)

• • Protocol for high-copy plasmids

• • elution with 50 µl H₂O

• measuring concentration with NanoDrop:

Sample	concentration in ng/µl
mycN clone 1	400.6
mycN clone 2	343.0
mycN clone 3	395.4
mycN clone 4	618.0
mycN clone 5	551.1
mycC clone 1	519.7
mycC clone 2	453.3
mycC clone 3	631.5
mycC clone 4	689.5
mycC clone 5	897.3

Further tasks:

• test digestion for confirmation

• sending for sequencing

Cultures of pARW089 and pARW071 for HPLC-Analysis

Investigators:Katharina, Nadine, Nicole, Steffi

Materials:

• overnight cultures of pARW089 and pARW071 from 2011-09-04 (Katharina)

• LB medium

• Kan

Method:

• preparing one liquid culture for each construct with Kanamycine

• incubate for 6 hrs in 37°C shaker (190 rpm)

Further tasks:

• HPLC analysis

Oligo-Fillin for mdnA-Library 2 (repetition)

Time: 2011-09-05, 10:00

Investigators: Nadine, Katharina, Jessica, Nicole, Steffi

Aim: Repetition of fillin reaction for library with new fillin program

Materials

• Primer, 25 µM :

• • # 76

• • # 74

• Klenow-Buffer 10X

• Klenow Fragment

• dNTPs

• water

Protocol:

• Reaction mix (50 µl)

• • 3 µl fw-Oligonucleotide (#76)

• • 3 µl rev-Oligonucleotide (#74)

• • 5 µl dNTPs (10 mM)

• • 5 µl Klenow-Buffer

• • 34 µl water

• • total volume: 50 µl
    

(2. Fillin program

• name: Fillin

• 3 min 94 °C

• 0.3°C per s (94°C-37°C)

• addition of 0.5 µl Klenow Fragment

• press enter

• 1hr 37°C)

Results:

• Output:

• mdnA-Lib2, Nad, 2011-9-5

Further tasks:

• digest w/ AatII

• ligation w/ pUP089 (digested w/ AatII and SfoI)

• transformation

Control restriction enzyme digestion of pSB1C3_mdnA_mycC and pSB1C3_mdnA_mycN

Investigators: Nadine, Nicole, Katharina, Steffi

Aim: Confirmation of the insertion of mdnA with myc-tag (N-terminal and C-terminal) in pSB1C3 vector

Time: 2011-09-05, 10:00-11:30

Material:

• clones:

• • pSB1C3_mdnA_mycC, clone 1, Katharina, 2011-09-05

• • pSB1C3_mdnA_mycC, clone 2, Katharina, 2011-09-05

• • pSB1C3_mdnA_mycC, clone 3, Katharina, 2011-09-05

• • pSB1C3_mdnA_mycC, clone 4, Katharina, 2011-09-05

• • pSB1C3_mdnA_mycC, clone 5, Katharina, 2011-09-05

• • pSB1C3_mdnA_mycN, clone 1, Katharina, 2011-09-05

• • pSB1C3_mdnA_mycN, clone 2, Katharina, 2011-09-05

• • pSB1C3_mdnA_mycN, clone 3, Katharina, 2011-09-05

• • pSB1C3_mdnA_mycN, clone 4, Katharina, 2011-09-05

• • pSB1C3_mdnA_mycN, clone 5, Katharina, 2011-09-05

• DraI (Fermentas)

• Tango buffer (Fermentas)

Method:

Reaction mix

• 2 µl DNA

• 2 µl Tango buffer

• 2 µl DraI (cuts three times)

• 14 µl H2O

Reaction conditions

• 37°C, 2 hours

Expected results:

• four fragments for each plasmid

• • fragment 1: 771 bases

• • fragment 2: 692 bases

• • fragment 3: 19 bases

• • fragment 4: 1114 bases

• pSB1C3_mdnA_mycC (four fragments)

• • fragment 1: 804 bases

• • fragment 2: 692 bases

• • fragment 3: 19 bases

• • fragment 4: 1082 bases

Conclusion:

Output: clone_name, cell type, stored fridge/freezer/-80°C; model saved as name in folder

PCR: mdnB, mdnABCDE

Time: 2011-09-05

Investigators: Nicole, Nadja

Materials

• vector: pARW089 (8,6 ng/µl), pARW071 (6,3 ng/µl), pARW089_T7 (8,6 ng/µl),

• 1-Phusion HF Polymerase, NEB

• 1-Phusion HF Buffer

• 2-PFU- Ultra HF Polymerase, NEB

• 2-PFU- Ultra HF Buffer, NEB

• dNTPs

• water

• Primer:

#	Primer
58	pf_mdnB_EcoRI_NotI_XbaI 12.08.
81	pr_mdnB_SpeI_NotI_PstI_30.08.
84	pf_mdnABCDE89_EcoRI_NotI_XbaI
82	r_mdnABCDE_iGEM

Protocol:

• mdnB

• • 1 µl pARW089 (diluted 1:100)

• • 1 µl dNTPs (10 mM)

• • 2.5 µl Primer forward (10 µM)- 81

• • 2.5 µl Primer backward (10 µM)- 58

• • 10 µl Phusion buffer HF

• • 0.5 µl Phusion HF Ploymerase (2U/µl)

• • 32.5 µl water

• • total volume: 50 µl

• mdnABCDE

• • 1 µl pARW089, 1 µl pARW089_T7, 1,3 µl pARW071

• • 1 µl dNTPs (10 mM)

• • 1 µl 2-PFU- Ultra HF Polymerase, NEB

• • 5 µl 2-PFU- Ultra HF Buffer, NEB

• • 40 µl (pARW089 pARW089_T7) water, 39,7 µl (pARW071) water

• • 1 µl Primer forward (10 mM) - 84

• • 1 µl Primer backward (10 mM)- 82

2. PCR programs

• IGMDNBB for mdnB

• first steps: 10x

• second steps: 20x

Step	Temperature	Time
Hot Start	98°C	Hold
Initial denaturation	98°C	30 sec
Denaturation	98°C	10 s
Annealing	64°C	20 s
Elongation	72°C	25 s
DenaturationII	98°C	10 s
AnnealingII	72°C	20 s
ElongationII	72°C	30 s
Final Elongation	72°C	10 min

• IGMDNAB

• first steps: 10x

• second steps: 20x

Step	Temperature	Time
Hot Start	98°C	Hold
Initial denaturation	98°C	30 sec
Denaturation	98°C	10 s
Annealing	69°C	20 s
Elongation	72°C	2 min 10 s
DenaturationII	98°C	30 s
AnnealingII	72°C	10 s
ElongationII	72°C	2min 30 s
Final extension	72°C	10 min

Results:

• M: DNA ladder mix (Fermentas)

• 1: mdnABCDE from pARW089, exp. size: ~ 6500 bp

• 2: mdnABCDE+T7 from pARW089, exp. size: ~ 6500 bp

• 3: mdnABCDE from pARW071, exp. size: ~ 6500 bp

• 4: -

• 5: mdnB, exp. size: ~1000 bp

• Output:

• • • mdnB worked out

• • • mdnABCDE did not work out

Different overnight cultures

Investigators: Jessica, Nadja

Time: 2011-09-04, 18:00

Materials:

• glycerol stocks of pARW089 and pARW071

• plates of pSB1C3 + mdnBC (from 2011-09-04), pUP089 A and B, pSB1A3_Ara_YFP, pSB1A3_Lac_YFP, pSB1K3_Ara_CFP, pSB1K3_Lac_CFP, pSB1K3_Lac_YFP (from 2011-09-03)

• LB medium

• Kan, Amp, Cm

Method:

• number of tubes: 5x mdnBC, 4x pUP089, 1x for the others

• incubate over night in 37°C shaker (200 rpm)

Further tasks:

• pSB1C3 + mdnBC: test digest

• pUP089: miniprep and glycerol stocks?

• expression backbones: glycerol stocks, preculture for expression test

• pARW089 and pARW071: cultures for HPLC

Purification of phages containing pPDV089

Investigators: Sandrina, Sabine

Aim: control if geneIII-mdnA will be expressed on the phage

Method/Materials:

• after over night incubation:

• centrifuge precipitaed phages: 5000 x g/ 45 min

• discard supernatant

• centrifuge again, 5000 x g/ 5 min

• remove supernatant carefully

• take pellet in 1 ml TBS

• move in 1.5 ml Eppi

• centrifuge again: 17000 x g/10 min

• supernatant in a new Eppi with 200 µl PEG-NaCl (mix!)

• incubate on ice for 60 min

• centrifuge precipitated phages: 17.000 x g/ 10 min (4°C)

• resuspend pellet in 300 µl TBS

• centrifuge:17 000 x g/ 10 min (4°C)

• supernatant in a fresh eppi= purified phages

Further tasks:

• measure concentration of phages
  

Glycerol stocks of phages containing pPDV089

Investigators: Sandrina

Aim: control if geneIII-mdnA will be expressed on the phage

Method/Materials:

• 300 µl phages in TBS

• 750 µl 86 % glycerol

• store at -80°C

Further tasks:

• measure concentration of phages
  

Concentration of phages containing pPDV089 measured by nanodrop

Investigators: Sandrina

Aim: control if geneIII-mdnA will be expressed on the phage

Results:

• OD 269: 0,095

• OD 320: 0,003

• phages per ml:

Ph/ml = (Abs 269- Abs 320) * 6*10^16/ bp (phagemid)

• bp (phagemid) = 10945

Ph/ml= 5*10^11

Further tasks:

• measure concentration of phages by serial dilution
  

87th Labday 2011-09-06

Glycerol stocks of pSB1C3_mdnBC and pUP089

Investigators: Nadine, Nicole, Niels

Aim: Preparing of glycerol stocks of pSB1C3_mdnBC and pUP089 for further use

Time: 2011-09-06, 8:30-9:15

Material:

Clones:

• • pSB1C3_mdnBC, clone A, source, date

• • pSB1C3_mdnBC, clone B, source, date

• • pSB1C3_mdnBC, clone C

• • pSB1C3_mdnBC, clone D

• • pSB1C3_mdnBC, clone E

• • pUP089 A clone A

• • pUP089 A clone B

• • pUP089 B clone A

• • pUP089 B clone B

Glycerol

Method:

• adding of each 700 µl over night culture to 300 µl glycerol

• gently inverting

Results:

• glycerol stocks

Output:

stored in freezer (-21°C); glycerolstock box

Number	Description	Strain	Resistance
G34	pSB1C3_mdnBC clone A	XL1-blue	cm
G35	pSB1C3_mdnBC clone B	XL1-blue	cm
G36	pSB1C3_mdnBC clone C	XL1-blue	cm
G37	pSB1C3_mdnBC clone D	XL1-blue	cm
G38	pSB1C3_mdnBC clone E	XL1-blue	cm
G39	pUP089 A clone A	XL1-blue	kana
G40	pUP089 A clone B	XL1-blue	kana
G41	pUP089 B clone A	XL1-blue	kana
G42	pUP089 B clone B	XL1-blue	kana

---

Miniprep of overnight cultures of pUP089 & psB1C3 + mdnBC

Investigators: Niels, Steffi

Materials:

• liquid culture of pSB1C3+ mdnBC & puP089

• NucleoSpin® Plasmid (NoLid) (Macherey-Nagel)

• • Protocol for high-copy plasmids

• • elution with 50 µl H₂O

• measuring concentration with NanoDrop:

Sample	concentration in ng/µl
puP089 A a	508 ng/µl
puP089 A b	504 ng/µl
puP089 B a	305 ng/µl
puP089 B b	602 ng/µl
psB1C3 + mdnBC a	444 ng/µl
psB1C3 + mdnBC b	665 ng/µl
psB1C3 + mdnBC c	661 ng/µl
psB1C3 + mdnBC d	625 ng/µl
psB1C3 + mdnBC e	609 ng/µl

Further tasks:

• test digestion for confirmation

• sending for sequencing

check plates - resistance of competent E. coli ER2738 cells

Investigators: Niels, Katharina, Sandrina, Steffi

Aim: check resistance of competent cells – ER2738 (from 2011-09-05, Niels, Katharina, Sandrina, Steffi)

Materials:

• agar plates from competent cells - E. coli ER2738 from 2011-09-05 (San/ Ste)
  

Results:

• all competent cells - E. coli ER2738 grow on agar plates with Tetracycline and without antibiotics

• no E. coli ER2738 clones on agar plates with Ampicillin, Kanamycine, Chloramphenicol

Conclusions:

• competent cells - E. coli ER2738 work and can be used for transformation

PCR mdnABCDE w/ Long PCR Enzyme mix from Fermentas

Time: 2011-09-06

Investigators: Nicole, Nadine

Aim: generate BioBrick of mdnABCDE, amplify mdnABCDE for ligation in pSB1C3

Materials

• vector: pARW089 (8,6 ng/µl), pARW071 (6,3 ng/µl)

Materials:

• DNA polymerases: Long PCR Enzyme Mix (Fermentas)

• Template DNA: pARW071 (vector)

• dNTPs

• water

• Primer:

#	Primer
84	pf_mdnABCDE89_EcoRI_NotI_XbaI (froward for pARW071 and pARW089)
82	r_mdnABCDE_iGEM (reverse for all)
83	pf_mdnABCDE89+T7_EcoRI_NotI_XbaI (froward for pARW089, generating mdnABCDE+T7)

Protocol:

• mdnABCDE

• • 2 µl vector

• • 1 µl dNTPs (10 mM)

• • 3 µl forward Primer

• • 3 µl reverse Primer

• • 0.3 µl Polymerase

• • 5 µl Buffer Fermentas Long Enzyme Mix (+MgCl2)

• • 35.3 µl water

• • total volume: 50 µl

2. PCR program

• IGMDNBB for mdnB

• first steps: 10x

• second steps: 20x

Step	Temperature	Time
Hot Start	94°C	Hold
Initial denaturation	94°C	180 sec
Denaturation	94°C	15 s
Annealing	59	30 s
Elongation	68°C	300 s
DenaturationII	94°C	15 s
AnnealingII	72°C	30 s
ElongationII	72°C	300 s + 2 s per each cycle s
Final Elongation	68°C	600 s

Results:

• Output:

• • mdnABCDE from pARW071, Nad/Nic, 6.9.11 (lane 2)

• • mdnABCDE from pARW089, Nad/Nic, 6.9.11 (lane 3)

• • mdnABCDE+T7 from pARW089, Nad/Nic, 6.9.11 (lane 4)

Further tasks:

• PCR purification

• digest

• ligation

PCR purification of mdnB

Investigators: Nadine, Nicole

Aim: Purification of mdnB (PCR product)

Time: 2011-09-06, 9:30-10:30

Material:

• PCR product of mdnB, PCR 2011-09-06, Nadja, Nicole, Jessica

• Machery-Nagel Nucleo Spin Extract II, PCR purification protocol

Method:

• done as described by manufacturer's protocol

• elution buffer

• elution volume 50 µl

Results:

• purified PCR product of mdnB

• cDNA = 44.6 ng/ µl

Output:

• stored in fridge, named: PCR product mdnB, pur., 2011-09-06

Further tasks:

• restriction enzyme digestion using SpeI and EcoRI

• Ligation in pSB1C3 (also digested with SpeI and EcoRI)

• Transformation in XL1-blue

Digest of purified PCR product mdnB and pSB1C3

Investigators: Niels

Materials:

• PCR product mdnB from 2011-9-5, Nad

• pSB1C3+mdnBC (a, b, c, d, e)

backseat driver asks: why do you digest our almost finished biobricks (pSB1C3 carrying mdnBC)? Why don't you use the vector pSB1C3 from the plasmid box?

• EcoRI, SpeI

• Buffer 4

• BSA

• H₂O

Protocol:

• PCR product:

• • 48 µl PCR product mdnB

• • 1 µl EcoRI

• • 1 µl SpeI

• • 6 µl Buffer 4

• • 0.6 µl BSA

• • 3.4 µl H₂O

• • total volume: 60µl

• pSB1C3+mdnBC:

• • 25 µl H₂O and DNA (see below)

• • 1 µl EcoRI

• • 1 µl SpeI

• • 3 µl Buffer 4

• • 0.3 µl BSA

• • total volume: 30µl

• pSB1C3+mdnBC dilutions

• • a: 9 µl vector + 16 water

• • b: 6 µl vector + 19 water

• • c: 6 µl vector + 19 water

• • d: 6.5 µl vector + 18.5 water

• • e: ´6.5 µl vector + 18.5 water

• incubation 5 hrs at 37°C

Output:

• PCR mdnB, pur., dig., Nie, 6.9.11

• pSB1C3+mdnBC a, dig., Nie, 6.9.11

• pSB1C3+mdnBC b, dig., Nie, 6.9.11

• pSB1C3+mdnBC c, dig., Nie, 6.9.11

• pSB1C3+mdnBC d, dig., Nie, 6.9.11

• pSB1C3+mdnBC e, dig., Nie, 6.9.11

Further tasks:

• purification

• ligation

PCR: Agarose Gel: mdnB digest verification; pSBIC3_B digest verification and PCR mdnABCDE with Long Enzyme Mix verification

Time: 2011-09-06

Investigators:Nadja

Materials

• digested mdnB

• digested pSBIC3_B

• PCR outcome of mdnABCDE (89, T7, 71)

Production of one 0,8 % agarose gel

• 0,8 % gel: 0.4 g agarose in 50 ml 1x TAE buffer

• Adding 2 µl gel red to the gel

Loading gels and running

Loading of gels

lane	Sample	Volume in µl	Expected size in bp
M	Gene Ruler DNA Ladder Mix	10µl (1:100)
1	71	2+ 3H2O+ 1 6x loading dye	6600
2	T7	2+ 3H2O+ 1 6x loading dye	6600
3	89	2+ 3H2O+ 1 6x loading dye	6600
4	mdnB	10+1 6x loading dye	1031
5	pSB1C3_B	30+ 6µl loading dye	2390, 243

Results:

Photos are already assigned to their appropriate experiments

Conclusions:

• test digest seems to be correct for mdnb (line 4), also the PCR was successful (line 1,2,3), only the pSB1C3_B digest did not worked out (line 5)

Further task:

• pSB1C3_A, C,D,E digest verification

PCR purification of PCR fragment mdnABCDE (T7, 89) and digested mdnB

Time: 2011-09-06

Investigators: Jessica, Nicole

Materials

• mdnABCDE+T7 from pARW089, Nad/Nic, 6.9.11

• mdnABCDE from pARW089, Nad/Nic, 6.9.11

• PCR mdnB, pur., dig., Nie, 6.9.11

• Macherey-Nagel Nucleo Spin Extract II kit
  

Protocol

• Protocol for PCR clean-up

• • elution with 35 µl NE buffer (incubation for 2 min at 70°C before centrifuging)

Output:

• mdnABCDE+T7 from pARW089, pur., Nadja/Jessica, 6.9.11

• mdnABCDE from pARW089, pur., Nadja/Jessica, 6.9.11

• PCR mdnB, pur., dig., pur., Nadja, 6.9.11 (concentration: 65.7 ng/µl)

NOTE: mdnABCDE from pARW071, Nad/Nic, 6.9.11 and the library were mixed up, so PCR and fillin reaction were repeated

Further task:

• digest of mdnABCDE+T7 from pARW089, pur., Nadja/Jessica, 6.9.11 and mdnABCDE from pARW089, pur., Nadja/Jessica, 6.9.11 for ligation with pSB1C3

• ligation of PCR mdnB, pur., dig., pur., Nadja, 6.9.11 with pSB1C3

PCR: Agarose Gel: pSBIC3_A, C, D, E

Time: 2011-09-06

Investigators:Nadja

Materials

• digested pSBIC3_ACDE

do you mean pSB1C3+mdnBC clone a to e? please use the same label names

Production of one 0,8 % agarose gel

• 0,8 % gel: 0.4 g agarose in 50 ml 1x TAE buffer

• Adding 2 µl gel red to the gel

Loading gels and running

Loading of gels

lane	Sample	Volume in µl	Expected size in bp
M	Gene Ruler DNA Ladder Mix	10µl (1:100)
1	pSB1C3_A	30+ 6x loading dye	2390, 243
2	pSB1C3_C	30+ 6x loading dye	2390, 243
3	pSB1C3_D	30+ 6x loading dye	2390, 243
4	pSB1C3_E	30+ 6x loading dye	2390, 243

Results:

300px

Conclusions:

• worked out

Further task:

• take pSB1C3_E for gel extraction and ligate it with mdnB

Gel Extraction of pSB1C3_E

Time: 2011-09-06

Investigators: Jessica, Nadja

Materials

• pSBIC3_E

• use of Macherey Nagel Nucleo Spin Extrac II kit
  

Protocol

• 100mg gel – 200µl NT buffer ---- incubate for 5-10min at 50 C

• load sample on colum and spin for 1min at 11000 x g

• add 700µl NT3 and spin for 1min at 11000 x g

• spin again for 2 min at 11000 x g

• eluate DNA with 35µl NE by incubating 2 min at 50 C and spinning down for 1 min at 11000 x g

Results:

• measured concentration: 24,8 ng/ µl

Further task:

• ligation with mdnB

Ligation of pSB1C3_E and mdnB

Time: 2011-09-06

Investigators: Jessica, Nadja

Materials

• pSBIC3_E

• mdnB

• use Ligation Calculator
  

Protocol

Components

Component	Molar Ratio	Concentration in ng/ µl	Length in bp	Max volume
Backbone	1	24,8	2390	7
Insert	3	1031	65,7	7

Ligation mix

• 1µl Buffer

• 1µl Enzyme

• 2,6 µl Insert

• 5,4 µl Backbone

• 0 µl water

• • total volume 10 µl

• • at 14 C over night

Further task:

• Transformation

prepare samples for sequencing (created pARWIII)

Investigators: Sandrina, Sabine

Aim: sequencing to control ligation of geneIII into pARW089(GATC)

Material/Method:

• purified plasmids of clones 1, 2 and 4) (70 ng/µl, 20 µl total volume)

• primer mdnA_6 (10 pmol/µl, 30 µl total volume)

Further tasks: control sequence

Digestion of pSB1C3

Investigators: Sandrina, Sabine

Aim:

• cloning of biobricks mdnA, geneIII and mdna/geneIII (fusion gene) into pSB1C3

Materials/Methods:

• 8 µl pSB1C3 (ca 2 µg)

• 2 µl NEB 10x buffer 2

• 1 µl restriction enzyme XbaI

• 1 µ restriction enzyme PstI

• 0,2 µl BSA

• 7,8 µl water

• 3 h at 37°C

Further tasks:

• gel electrophoresis for purification

• ligation of mdnA, geneIII and mdna/geneIII (fusion gene) into pSB1C3

digestion of vector pARWIII

Investigators: Sandrina, Sabine

Aim: deletion of kanamycin gene in pARWIII (pARW089 containing geneIII

Materials/Methods:

• 2 µg sample (3 clones of pARWIII)

• 2 µl NEB 10x buffer 3

• 1 µl restriction enzyme NsiI

• add water: total volume 20 µl

• 3 h at 37°C

Further tasks:

• gel electrophoresis for purification

• re-ligation

Digestion of PCR products geneIII and mdnA for cloning into pSB1C3

Investigator: Sandrina, Sabine

Aim: cloning of mdnA, geneIII and mdnA/geneIII into pSB1C3

Material/Method:

• 30 µl PCR product mdnA

• 1 µl restriction enzyme XbaI

• 1 µl restriction enzyme PstI

• 4 µl NEB 10x buffer 2

• 0,4 µl BSA

• 3,6 µl water

• 1,5 h, 37°C

• 20 µl PCR product geneIII

• 1 µl restriction enzyme XbaI

• 1 µl restriction enzyme PstI

• 3 µl NEB 10x buffer 2

• 4,7 µl water

• 0,3 µl BSA

• 1,5 h, 37°C

• 20 µl PCR product geneIII

• 1 µl restriction enzyme NgoMIV

• 1 µl restriction enzyme PstI

• 3 µl NEB 10x buffer 1

• 0,3 µl BSA

• 4,7 µl water

• 1,5 h, 37°C

• 20 µl PCR product mdnA

• 1 µl restriction enzyme XbaI

• 1 µl restriction enzyme AgeI

• 3 µl NEB 10x buffer 1

• 4,7 µl water

• 0,3 µl BSA

• 1,5 h, 37°C

Further Tasks:

• gel electrophoresis for purification

• ligation of mdnA and geneIII into pSB1C3

Agarose gel electrophoresis for purification of digested PCR products and vectors

Investigator: Sandrina, Sabine

Aim: control and purification of digested PCR products mdnA and geneIII

Material/Method:

• digested PCR products (mdnA and geneIII)

• digested vectors (pSB1C3 and pARWIII)

• 1 % agarose gel

• 1x TAE buffer

• Gel Red

• DNA Ladder Mix (1:10) (Fermentas)

• 6x Loading Dye (Fermentas)

• Gel Red

Ligation of geneIII and mdnA into pSB1C3

Investigator: Sabine

Aim: pSB1C3 containing mdnA, geneIII and mdnA/geneIII

Material/Method:

• 4,5 µl insert mdnA (7 ng/µ)

• 10 µl pSB1C3 (10 ng/µl)

• 2 µl T4 ligase buffer (Fermentas)

• 1 µl T4 Ligase (Fermentas)

• 2,5 µl water

• 1 h at room temperature

• 6,3 µl insert geneIII (10 µg/µl)

• 10 µl pSB1C3 (10 ng/µl)

• 1 µl T4 ligase buffer (Fermentas)

• 2 µl T4 Ligase (Fermentas)

• 0,8 µl water

• 1 h at room temperature

• 3,2 µl insert mdnA (7,6 µ/µl)

• 6,5 µl insert geneIII (7 µg/µl)

• 7,3 µl pSB1C3 (10 ng/µl)

• 2 µl T4 ligase buffer (Fermentas)

• 1 µl T4 Ligase (Fermentas)

• 1 h at room temperature

Further task: transformation

Re-ligation of NsiI-digested pARWIII

Investigator: Sabine

Aim: create pARW089 containing geneIII without kanamycin resistence (for library)

Material/Method:

• 17 µl NsiI-digested pARWIII (clones 1, 2 and 3)

• 1 µl T4 ligase buffer (Fermentas)

• 2 µl T4 Ligase (Fermentas)

• 1 h at room temperature

Further task: transformation

Transformation of created vectors in E. coli

Investigator: Sabine

Aim:amplification of vectors

Material:

• pARWIII without kanamycin resistence (clones 1, 2 and 3)

• pSB1C3 containing mdnA, geneIII or mdnA/geneIII

• agar plates containing chloramphenicol (pSB1C3)

• agar plates containing kanamycin, ampicillin(pPDV)

• agar plates containing kanamycin (pARW089)

Method:

• addition of 4 µl ligation reaction to XL1-blue cells
  

• incubation 25 min on ice,
  

• heat shock 45 sec at 42°C,
  

• incubation 2 min on ice,
  

• addition of 750 µl LB medium,
  

• incubation 60 min at 37 °C and 750 rpm
  

• plating on agar plates containing 100 µg/ml tetracyclin and 100 µg/µl ampicillin
  

• storage over night at 37°C
  

Further tasks:
control cell clones

PCR of TorA-bla with clevage site of 14_3C and TEV for BioBricks

For better understanding of described experiment see also: http://141.89.201.101/iGEM/wiki2011/images/c/c8/UP_SG_Klonierungsschema_Protease.pptx

Investigators: Sascha, Stefan, Sebastian

Aim: get PCR fragment of TOR bla sequence for BioBricks

Methode:

Primer TEV:

(1)

(2)

Methode:

PCR

• Template: 1 µL (pJC354 <10ng)

• Nucleotides: 1 µL of 10 mM ready to use dNTP mix
  

• 5 µL 10 x Amplification buffer S
  

• 2 µL 25 mM MgCl2
  

• 2,5 µL primers = 25 pmol absolute (2,5 µL of each primer)
  

• 35,5 µL of pure water
  

• 0,5 µL TaqPol
  

Program:

• Denat: 4 min 94°C

• 5x:

Denat: 1 min 94°C

Anneal: 1 min 51°C

Extend: 1 min 72°C

• 25x:

Denat: 1 min 94°C

Anneal: 1 min 61°C

Extend: 1 min 72°C

• Final Extend: 10min 72°C

Further Tasks:

PCR purification

PCR: mdnABCDE

Time: 2011-09-06

Investigators: Nicole, Nadja

Materials

• vector: pARW089 (2*)

• Long Enzyme Mix (+MgCl2) Buffer Fermentas

• Long Enzyme Polymerase Fermentas

• dNTPs (NEB)

• water

• Primer:

#	Primer
x	pf_mdnABCDE089_NotI_SpeI_30.08.
x	r_mdnABCDE_iGEM

Protocol:

• • 2 µl pARW089 (diluted 1:100)

• • 1 µl dNTPs (10 mM)

• • 3 µl Primer forward (10 µM)

• • 3 µl Primer backward (10 µM)

• • 5 µl buffer

• • 0.3 µl Ploymerase

• • 35,7 µl water

• • total volume: 50 µl

• mdnABCDE

2. PCR programs

• IGLONG2
  

Result

• PCR worked out

Cultures of pARW071 and pARW089 for HPLC

Time: 2011-09-06

Investigators: Nicole, Nadine, Jessica

Materials

• overnight cultures of pARW089 and pARW071

• 500mM Tris-HCl, pH 7.4

Protocol: (following protocol from Elke)

• inoculation of 400 ml LB medium with antibiotic

• at 37°C for about 7 h

• centrifuged 4x at 6000 g for 10 min in 50 ml falcons

• washed with Tris-HCl

Output:

• 4 falcons stored in -20°C (71 1, 71 2, 89 1, 89 2)

Overnight cultures of expression backbones

Time: 2011-09-06

Investigators: Jessica

Aim: Precultures for test of expression backbones

Materials

• plate of pSB1A3_Ara_YFP (from 2011-09-03)

• test cultures of pSB1A3_Lac_YFP, pSB1K3_Ara_CFP, pSB1K3_Lac_CFP (from 2011-09-06)

• LB medium, Kan, Amp

Method:

• inoculation of 10 ml LB medium with antibiotic

• overnight at 37°C

Further tasks:

• testing expression

88th Labday 2011-09-07

Digestion and Ligation of TorA-bla with cleavage site of 14_3C and TEV for BioBricks and TEV / 14_3C biobricks

For better understanding of described experiment see also: http://141.89.201.101/iGEM/wiki2011/images/c/c8/UP_SG_Klonierungsschema_Protease.pptx

Investigators: Sascha, Paul

Aim: purification and digestion PCR fragment (produced: 06.09.2011) of TOR bla sequence for BioBricks

Methode:

PCR-Products were purificated using Nucleospin Extract II KIT.

• The torA-bla construct was digested with AgeI and XbaI

• • The vector (BBa_K404304_pSB1C3) for torA-bla was digested with AgeI and XbaI

• 14_3C and Tev proteases were digested with EcoRI and SpeI

• • The vector (BBa_K404304_pSB1C3) for proteases was digested with EcoRI and SpeI

The digestion was allowed to proceed for 2h at 37°C.

• Digested products were resolved on 1% agarose gel, the corresponding bands were excissed and extracted from the gel using Nucleospin gel extraction KIT

• • Expected bands:

• • Tev: 760 bp (bands: Tev=2,Tev2=3,Tev4=4, Tev7=5)

• • 14_3C: 570 bp (bands: 14_3C1=15, 14_3C2=16)

• • TorA-bla: 1001 bp (bands: Cs_TevI=6, Cs_TevII=7, Cs_TevIII=8, Cs_14_3CI=10, Cs_14_3CII=11)

• • Vector: ~2632 bp (bands: Vector for proteases=14, vector for Tor_bla=17)

400px 400px

• The excised fragments were ligated into corresponding digested and excised vectors: Tev2, Tev3, Tev4, Tev7 and 14_3C1 and 14_3C2 into vector for proteases; the torA-bla fragments were ligated into vector for TorA-bla)

• ligation was calculated with gibthon ligation calculator ([http://www.gibthon.org/ligate.html Klick]) with a molar ration of 1:3 of plasmid to insert.

Proteases:

• 1 µl T4-ligation (Fermentas)

• 1 µl Ligase Buffer

1.4 µl vector - 6.6 µl Tev

1.2 µl vector - 6.8 µl Tev2

0.7 µl vector - 7.3 µl Tev4

1.5 µl vector - 6.5 µl Tev7

0.3 µl vector - 7.7 µl 14_3C1

0.4 µl vector - 7.6 µl 14_3C2

• = 6x10 µl ligation batches

TorA-bla

• 1 µl T4-ligation (Fermentas)

• 1 µl Ligase Buffer

2.2 µl vector - 5.8 µl Cs_14_3CI

2.4 µl vector - 2.6 µl Cs_14_3CII

1.5 µl vector - 6.5 µl Cs_TevI

1.7 µl vector - 6.3 µl Cs_TevII

2.1 µl vector - 5.9 µl Cs_TevIII

• = 5x10 µl ligation batches

• ligation was allowed to proceed for 1h at room temperature.

• 2µl of each ligation batch was transformed into competent Xl1-blue cells.

• • Cells were plated in Cm containing agar plates

Oligo-Fillin for mdnA-Library (repetition)2

Time: 2011-09-07, 7:20-8:40

Investigators: Nadine, Nicole

Aim: Production of a library carrying mutated mdnA ß-lactamase-screening (carrying AatII restriction site)

Material:

• oligos (25 mM, HPLC purified, ordered by Sigma Aldrich):

• • o_mdnA_library, 76

• • o_focused_library_2, 74

• 10x Klenow buffer

• dNTPs (10 mM)

• H2O

Method:

• Reaction mix (total volume: 50 µl)

• • 3 µl o_mdnA-library, 76

• • 3 µl o_focused_library_2, 74

• • 5 µl dNTPs

• • 5 µl Klenow buffer

• • 34 µl H2O

• Reaction conditions

• • Program: ORIGAMI1

• after finishing program ORIGAMI1:

• • addition of 1 µl Klenow-fragment

• • incubation 1 hour, 37°C

Results:

• hybridized oligo for production of mdnA-library

Output:

• hybridization product, named foc 2

• stored in thermocycler

Further tasks:

• PCR purification

• restriction enzyme digestion using SfoI and AatII, also of pUP089

Preparing samples mdnC, mdnD, mdnE and mdnABC for sequencing

Time: 2011-09-07,

Investigators: Katharina, Nadine, Nicole

Purification of Fillin mdnA-Lib2

Time: 2011-9-7,

Investigators: Nadine, Nicole

Aim: Purification of oligos for focused library 2

Materials

• product of filled-in reaction, 2011-09-07, Nadine, Nicole

• Machery-Nagel Nucleo Spin Extract II kit

Method:

• application based on manufacturer’s protocol for PCR product purification

• exceptions for elution procedure:

• • H2O used for elution

• • elution volume V=25 µl

• • before centrifugation (for 1 min) incubation 5 min at 70°C

• DNA concentration measured by Nanotrop

Results:

• cDNA: 113.3 ng/ µl

Output:

• purified oligos (focused library 2)

Further tasks:

• digestion using SfoI and AatII

• digestion of pUP089 using SfoI and AatII

• Purification of both

• Ligation of pUP089 and oligos (focused library 2)

digest of purified Fillin mdnA-Lib2 and pUP089

Time: 2011-9-7,

Investigators: Nadine, Nicole

purification of digested pSB1C3 and pUP089

Time: 2011-09-07,

Investigators: Niels, Jessica, Katharina

Ligation of Fillin mdnA-Lib2 and pUP089

Time: 2011-9-7,

Investigators: Nadja

Materials

• product of digested and purified Fillin mdnA Lib2 (2011-9-7, Nicole, Nadine) = 123,0 ng/ µl

• pUP089 = 14,9 ng/ µl

• Quick Ligase Buffer

Protocol:total volume of 20µl+

backseat driver asks: where is the ligation control? we talked about this several times!!!!!!!!! now we have to repeat this -.- and time designation would be also nice

• 10 µl Quick ligase buffer

• 1µl Enzyme

• 1µl Insert

• 8µl backbone

• • at 16 °C in PCR machine

Further tasks:

• transformation

Ligation of mdnABCDE in pSB1C3

Time: 2011-9-7,

Digest of purified PCR product mdnABCDE and pSB1C3

Time: 2011-9-7, 8:30-12:15

Investigators: Nadine, Nicole

Aim: generate a BioBrick of mdnABCDE, digest for ligation in pSB1C3

Materials:

• PCR products:

• • mdnABCDE from pARW089 from 2011-9-6, Nad/Nic

• • mdnABCDE+T7 pARW089 from 2011-9-6, Nad/Nic

• • mdnABCDE from pARW089 or pARW071 from 2011-9-6, Nadj

• BBa_K404304_pSB1C3 (#3), 284.6 ng/µl

• EcoRI, SpeI

• Buffer 4

• BSA

• H₂O

Protocol:

• PCR product:

• • 30 µl PCR product

• • 1 µl EcoRI

• • 1 µl SpeI

• • 5 µl Buffer 4

• • 0.6 µl BSA

• • 12.5 µl H₂O

• • total volume: 50µl

• vector:

• • 5 µl vector

• • 1 µl EcoRI

• • 1 µl SpeI

• • 2 µl Buffer 4

• • 0.2 µl BSA

• • 10.8 µl H₂O

• • total volume: 20µl

• incubation 3 hrs at 37°C (start: 9:15)

Output:

• mdnABCDE from pARW089, Eco, Spe, from 2011-9-7, Nad/Nic

• mdnABCDE+T7 pARW089, Eco, Spe from 2011-9-7, Nad/Nic

• mdnABCDE from pARW089, Eco, Spe or pARW071 from 2011-9-7, Nad/Nic

• BBa_K404304_pSB1C3, Eco, Spe, from 2011-9-7, Nad/Nic

Further tasks:

• agarose gel

• purification

• ligation

test digest of mdnBC

Time: 2011-9-7,10:30-

Investigators: Nadine, Nicole

Aim: prove of Insert (mdnBC)

Materials:

• mdnBC klon a-e, Nie, 6.9.11

• HindIII, AvaI, (NEB)

• Buffer 2 (NEB, 10x)

• water

Plan:

insert	plasmid size in bp	enzymes	buffer	Expected size in bp
mdnBC	4419	HindIII, AvaI	2	363, 836, 3220

 Digestion protocol 

• 1 µl DNA

• 0.5 µl HinIII

• 0.5 µl AvaI

• 2 µl 10x buffer

• 16 µl pure water

• total: 20µl

• 37°C for 2 hrs

Conclusions:

Further tasks:

• agarose gel

• sequencing

test pSB1A3_Ara_YFP and pSB1A3_Lac_YFP (expression backbones)

Time: 2011-9-7,12

Investigators: Nadine, Nicole, Jessica

Method:

• pSB1A3_Lac_YFP:

• • ODs at 16:45: 0.095 (1) and 0.082 (2, control)

• • no induction

• pSB1A3_Ara_YFP:

• • ODs at 17:30 : 0.657 (1) and 0.713 (2, control)

• • induction with arabinose, end concentration 5 mM

Results:

• fluorescence didn't increase, curve flattened

PCR: mdnABCDE

Time: 2011-09-06

Investigators: Nadja

Materials

• vector: pARW071 (1)

• Long Enzyme Mix (+MgCl2) Buffer Fermentas

• Long Enzyme Mix Polymerase Fermentas

• dNTPs (NEB)

• water

• Primer:

#	Primer
84	pf_mdnABCDE089_NotI_SpeI_30.08.
82	r_mdnABCDE_iGEM

Protocol:

• 2,6 µl pARW08 (diluted 1:100)

• 1 µl dNTPs (10 mM)

• 3 µl Primer forward (10 µM)

• 3 µl Primer backward (10 µM)

• 5 µl buffer

• 0.3 µl Ploymerase

• 35,4 µl water

• • total volume: 50 µl

• mdnABCDE

2. PCR programs

• IGLONG2
  

Further task

• gel verification

• purification

Overnight cultures of expression backbones and pUP089

Time: 2011-09-07, 18:00

Investigators: Niels

Aim: Precultures for test of expression backbones and glycerol stocks

Materials

• plates of pSB1A3_Ara_YFP, pSB1A3_Lac_YFP, pSB1K3_Ara_CFP, pSB1K3_Lac_CFP (from 2011-09-03)

• plates of pUP089 A and B

• LB medium, Kan, Amp

Method:

• inoculation of 10/5 ml LB medium with antibiotic

• overnight at 37°C

Further tasks:

• testing expression

• glycerol stocks !!!!!!!

Repeated test digest of mdnBC

Time: 2011-9-7, 20:00

Investigators: Jessica, Niels, Nadja

Aim: prove of Insert (mdnBC)

Materials:

• mdnBC clone b, Nie, 6.9.11

• HindIII, AvaI, PstI-HF, XmaI (NEB)

• Buffer 2 and 4 (NEB, 10x)

• water

Plan:

insert	plasmid size in bp	enzymes	buffer	Expected size in bp
mdnBC	4419	HindIII, AvaI	2	363, 836, 3220
mdnBC	4419	PstI-HF, XmaI	4	1559, 2868

Digestion protocol

• 1 µl DNA

• 0.5 µl per enzyme

• 2 µl 10x buffer

• 16 µl pure water

• total: 20µl

• 37°C for overnight (on thermomixer in iGEM lab)

Agarose gel:

Conclusions:

Further tasks:

• sequencing

Production of phages containing pPDV089 in ER2738 cells

Investigators: Sabine, Sandrina, Steffi

Aim: control if geneIII-mdnA will be expressed on the phage

Method/Materials:

• first step: amplification of cells containing pPDV089 (clone: 2S14):

• 50 ml DYT medium will be inoculated with the cells, so that OD600 = 0.1

• add antibiotics tetracyclin and ampicillin to the medium

• cells should be incubated 37 °C till OD600 = 0.3-0.5 (here: 0.356) is reached

• second step: infection with helper phages

• add helper phages 10^11 phages/50 ml (3,5 µl)

• incubate for 10 min at 37°C (without shaking!)

• add 0,5 mM IPTG

• incubate 50 min at 28°C and rpm

• add 70 µg/ml kanamycin and incubate for 5 h at 28°C (...)

• third step: phage purification

• centrifuge cell culture at 5000 x g/ 15 min

• fill supernatant in a new 50 ml falcon and centrifuge again (5000 g/15 min)

• 40 ml of the supernatant with 8 ml PEG-NaCl (20% (w/v) PEG-8000, 2,5 M NaCl)

• incubate over night at 4°C

Further tasks:

• go on with phage purification
  

SDS- PAGE for Western Blotting and coomassie staining of purified phages

Investigators: Sabine, Sandrina, Sebastian

Aim: control if geneIII-mdnA is expressed on the phages produced in XL1-blue cells

Method/Materials:

Further tasks:

• Western Blot and coomassie staining
  

Western Blot

Investigators: Sabine, Sandrina

Aim: control if geneIII-mdnA is expressed on the phages produced in XL1-blue cells

Method/Materials:

• membrane impregnated in methanol

• whatman paper

• blotting buffer

• blotting chamber

• 1 h at 200 mA

• blocking over night in TBS with 5% milk powder

Further tasks:

• antibody detection
  

Coomassie staining

Investigators: Sabine, Sandrina

Aim: control if geneIII-mdnA is expressed on the phages produced in XL1-blue cells

Method/Materials:

• 20 ml water

• 20 ml methanol

• 60 ml coomassie stock solution

• gel storage over night in coomassie solution shaking
  

Further tasks:

• destaining of the gel
  

overnight culture of picked E. coli clones transformed with pARWIII (pARW089 containing geneIII) to control deletion of kanamycin gene, pSB1C3 containing mdnA, geneIII or mdnA/geneIII

Investigators: Sandrina, Sabine

Aim: control deletion of kanamycin gene in created pARWIII, control ligation of mdnA, geneIII and mdnA/geneIII into pSB1C3

Method/Materials:

• 5 clones from pARW089 with geneIII (kanamycin)

• 5 clones from pSB1C3 with mdnA (chloramphenicol)

• 5 clones from pSB1C3 with geneIII (chloramphenicol)

• 5 clones from pSB1C3 with mdnA/geneIII (chloramphenicol)

• 5 ml LB medium per clone

• storage over night at 37°C and 800 rpm
  

Further tasks:

• test digestion
  

89th Labday 2011-09-08

Oligo-Fillin for mdnA-library 2 (repetition) and for Phage-display-mdnA-library (carrying AgeI restriction site)

Investigators: Nicole, Nadine

Time: 2011-09-08, 11:00-12:30

Aim: Production of libraries carrying mutated mdnA, one for phage-display (carrying ageI restriction site) and one for ß-lactamase-screening (carrying AatII restriction site)

Material:

• oligos (25 mM, HPLC purified, ordered by Sigma Aldrich):

• • o_mdnA_library, 76 à for both libraries

• • o_focused_library_2, 74 à for AatII carrying library

• • o_focused_library_AgeI, à for AgeI carrying library

• 10x Klenow buffer

• dNTPs (10 mM)

• H2O

Method:

• Reaction mix (total volume: 50 µl)

• • 3 µl fw-oligonucleotide (o_mdnA-library, 76)

• • 3 µl rev-oligonucleotide (either o_focused_library_2, 74 or o_focused_library_AgeI, )

• • 5 µl dNTPs

• • 5 µl Klenow buffer

• • 34 µl H2O

• Reaction conditions

• • Program: ORIGAMI1

• after finishing program ORIGAMI1:

• • addition of 1 µl Klenow-fragment

• • incubation 1 hour, 37°C

Results:

• hybridized oligos for production of mdnA-library

Output:

• two hybridization products

• • 1) AgeI-library, named AgeI

• • 2) AatII-library, named foc 2

• stored in thermocycler

Further tasks:

• PCR purification

• restriction enzyme digestion using SfoI and either AatII or AgeI, also of either pUP089 or pARWIII

Transformation of XL1 with different constructs

Time: 2011-09-08, 12:30-15:00

Investigators: Nadine, Nicole

Materials:

• XL1-Blue

• ligation products:

• • pSB1C3+mdnABCDE+T7, Niels, 7.9.11, resistance: cm

• • pSB1C3+mdnABCDE from pARW089, Niels, 7.9.11, resistance: cm

• • pSB1C3+mdnB, Jes/Nadja, 6.9.11, resistance: cm

• • pSB1C3+H20 (control), Jes/Nadja, 6.9.11, resistance: cm

• • pUP089+lib2, Nadja, 7.9.11, resistance: kan

• plasmids from miniprep (Kat, 2011-8-20):

• • pSB1A3_Ara_YFP clone A (#1), resistance: amp

• • pSB1A3_Lac_YFP clone B (#4), resistance: amp

• • pSB1K3_Ara_CFP clone A (#25), resistance: kan

• • pSB1K3_Lac_CFP clone B (#21), resistance: kan

• • pSB1K3_Lac_YFP clone C (#18), resistance: kan
    

• antibiotics from stock:

• • kanamycin

• • ampiciline

• • chloramphenicol

• LB-Agar

Method:

• ligation products:

• • addition of 2 µl ligation reaction to cells (XL1-blue) in 1.5 ml Eppi,

• incubation 30 min on ice,

• heat shock 60 sec at 42°C,

• incubation 5 min on ice,

• addition of 750 µl LB medium,

• incubation at 37 °C shaking for 60 min,

• plating on LB medium with appropriate antibiotic (20 ml Agar and 20 µl antibiotic (see above) per plate)

• storage over night at 37°C (start: 15:00)

Further tasks:

• Picking clones for overnight culture (except for library 2)

• Producing glycerol stocks

• count colonies of pUP089_lib2 and collect them w/ LB

Output:

• Plates w/ XL1 blue:

• • pSB1C3+mdnABCDE+T7, Niels, 7.9.11, resistance: cm

• • pSB1C3+mdnABCDE from pARW089, Niels, 7.9.11, resistance: cm

• • pSB1C3+mdnB, Jes/Nadja, 6.9.11, resistance: cm

• • pSB1C3+H20 (control), Jes/Nadja, 6.9.11, resistance: cm

• • 10x pUP089+lib2, Nadja, 7.9.11, resistance: kan

• • pSB1A3_Ara_YFP clone A (#1), resistance: amp

• • pSB1A3_Lac_YFP clone B (#4), resistance: amp

• • pSB1K3_Ara_CFP clone A (#25), resistance: kan

• • pSB1K3_Lac_CFP clone B (#21), resistance: kan

• • pSB1K3_Lac_YFP clone C (#18), resistance: kan

• Ligation products stored in freezer, green box, mdn-BioBricks

• • pSB1C3+mdnABCDE+T7, pARW089, 7.9.11

• • pSB1C3+mdnABCDE, pARW089, 7.9.11

• • pSB1C3+mdnB, Nadja/Jes, 6.9.11

• • pSB1C3+H20, Nadja/Jes, 6.9.11

• Expresion backbones stored in white/colorless box w/ label: Expression Backbones 2

Purification of Fillin mdnA-Library_2 (carrying either AgeI or AatII restriction site) and PCR product of mdnABCDE from pARW071

Time: 2011-9-8,

Investigators: Nadine, Nicole

Aim: Purification of oligos for focused library 2 for phage display and ß-lactamase screening and purification of PCR product mdnABCDE from pARW071 for further use

Materials

• product of filled-in reaction, 2011-09-08, Nadine, Nicole

• • Age (mdnA library for phage display)

• • foc 2 (mdnA library for ß-lactamase screening)

• PCR product of mdnABCDE from pARW071 (named mdnA71), 2011-09-07, Nadja

• Machery-Nagel Nucleo Spin Extract II kit

Method:

• application based on manufacturer’s protocol for PCR product purification

• exceptions for elution procedure:

• • H2O used for elution

• • elution volume V=25 µl

• • before centrifugation (for 1 min) incubation 5 min at 70°C

• DNA concentration measured by Nanotrop

Output:

• purified oligos

• • mdnA focused library 2 (carrying AatII restriction site), named Age, stored in freezer

• • mdnA focused library 2 (carrying AgeI restriction site), named foc 2, stored in freezer

• • mdnABCDE from pARW071, named mdnA71, stored in freezer

Further tasks:

• digestion of foc 2 using SfoI and AatII

• digestion of pUP089 using SfoI and AatII

• digestion of Age using SfoI and AgeI

• digestion of pARWIII using SfoI and AgeI

• digestion of mdnA71 using EcoRI and SpeI

• digestion of pSB1C3 using EcoRI and SpeI

Restriction enzyme digestion of mdnA focused library 2 carrying AatII restriction site and pUP089

Investigators: Nicole, Nadine

Time: 2011-09-08,

Aim: Digest of hybridized oligos for further production of mdnA focused library

Material:

• clones

• • purified mdnA oligos carrying AatII restriction site, named foc 2, 2011-09-08, Nicole/Nadine

• • pUP089, clone A I, Nadine

• NEB buffer 4

• AatII

• SfoI

Method:

• Mix for digestion of foc 2

• • 23 µl PCR purification product foc 2 (mdnA oligos)

• • 2 µl AatII

• • 2 µl SfoI

• • 3 µl NEB buffer 4

• Mix for digestion of pUP089

• • 4 µl DNA pUP089

• • 2 µl AatII

• • 2 µl SfoI

• • 2 µl NEB buffer 4

• • 10 µl H2O

• Incubation for 2 hours, 37 °C

Results:

• digested oligos for library

• digested pUP089

Output:

• digested oligos for mdnA library (carrying AatII restriction site), named foc 2, stored in ice box

• digested pUP089, named pUP089, stored in ice box

Further tasks:

• PCR purification (foc 2) resp. agrose gel and gel extraction (pUP089)

• ligation of both

Restriction enzyme digestion of mdnABCDE71 and pSB1C3

Investigators: Nicole, Nadine

Time: 2011-09-08,

Aim: Digest of PCR product mdnABCDE from pARW071 and pSB1C3 to prepare a biobrick

Material:

• clones

• • purified PCR product mdnABCDE, named mdnABCDE71, 2011-09-08, Nicole/Nadine

• • pSB1C3,

• NEB buffer 4

• EcoRI-HF

• SpeI

• BSA

Method:

• Mix for digestion of mdnABCDE from pARW071 (total volume 50 µl)

• • 23 µl PCR purification product foc 2 (mdnA oligos)

• • 1 µl EcoRI-HF

• • 1 µl SpeI

• • 5 µl NEB buffer 4

• • 0.6 µl BSA

• • 19.5 H2O

• Mix for digestion of pSB1C3 (total volume 20 µl)

• • 5.8 µl DNA pSB1C3

• • 1 µl EcoRI-HF

• • 1 µl SpeI

• • 2 µl NEB buffer 4

• • 10 µl H2O

• • 0.2 µl BSA

• Incubation for 2 hours, 37 °C

Results:

• digested oligos for library

• digested pARWIII

Output:

• digested mdnABCDE from pARW071, named digested mdnABCDE71, stored in ice box

• digested pSB1C3, named digested pSB1C3, stored in ice box

Further tasks:

• PCR purification (mdnABCDE71) resp. agrose gel and gel extraction (pSB1C3)

• ligation of both

Restriction enzyme digestion of mdnA focused library 2 carrying AgeI restriction site and pARWIII

Investigators: Nicole, Nadine

Time: 2011-09-08,

Aim: Digest of hybridized oligos for further production of mdnA focused library for phage-display

Material:

• clones

• • purified mdnA oligos carrying AgeI restriction site, named Age, 2011-09-08, Nicole/Nadine

• • pARWIII, clone 1_1, Sandrina, 2011-09-08

• NEB buffer 4

• AgeI

• SfoI

Method:

• Mix for digestion of Age

• • 22.5 µl PCR purification product foc 2 (mdnA oligos)

• • 2.5 µl AgeI

• • 2 µl SfoI

• • 3 µl NEB buffer 4

• Mix for digestion of pARWIII

• • 6.5 µl DNA pARWIII

• • 2.5 µl AgeI

• • 2 µl SfoI

• • 3 µl NEB buffer 4

• • 6 µl H2O

• Incubation for 2 hours, 37 °C

Results:

• digested oligos for library

• digested pARWIII

Output:

• digested oligos for mdnA library (carrying AgeI restriction site), named Age, stored in ice box

• digested pARWIII, named digested-pARWIII, stored in ice box

Further tasks:

• PCR purification (Age) resp. agrose gel and gel extraction (pARWIII)

• ligation of both

PCR of TorA-bla with clevage site of 14_3C and TEV for BioBricks

For better understanding of described experiment see also: http://141.89.201.101/iGEM/wiki2011/images/c/c8/UP_SG_Klonierungsschema_Protease.pptx

Investigators: Sascha, Stefan, Sebastian

Aim: get PCR fragment of TOR bla sequence for BioBricks

Methode:

Primer TEV:

(1)

(2)

Methode:

PCR

• Template: 1 µL (pJC354 <10ng)

• Nucleotides: 1 µL of 10 mM ready to use dNTP mix
  

• 10 µL 5 x Amplification buffer
  

• 2,5 µL primers = 25 pmol absolute (2,5 µL of each primer)
  

• 35 µL of pure water
  

• 0,5 µL Fusion
  

Program:

• Denat: 4 min 94°C

• 30x:

Denat: 30 sec 94°C

Anneal: 30 sec 72°C

Extend: 1 min 72°C

• Final Extend: 10min 72°C

Further Tasks:

PCR purification

Test digest of pSB1C3+mdnDE

Time: 2011-9-08, 17:30-20:30

Investigators: Jessica

Aim: prove of Insert (mdnDE)

Materials:

• pSB1C3+mdnBC clones K1-K6, Kat, 8.9.11

• • concentrations:

Sample	Concentration in ng/µl
clone K1	835.1
clone K2	775.6
clone K3	488.1
clone K4	639.3
clone K5	554.3
clone K6	551.5

• HpaI (NEB)

• Buffer 4 (NEB, 10x)

• water

Plan:

insert	plasmid size in bp	enzymes	buffer	Expected size in bp
mdnDE	5154	HpaI	4	1329, 3825

Digestion protocol

• 1 µl DNA

• 0.5 µl HpaI

• 2 µl 10x buffer

• 16 µl pure water

• total: 20µl

• 37°C for
  

Agarose gel

lane	Sample	Volume in µl
M	Gene Ruler DNA Ladder Mix (1:10)	20µl
1	-
2	clone K1	20 + 4x loading dye
3	clone K2	20 + 4x loading dye
4	clone K3	20 + 4x loading dye
5	clone K4	20 + 4x loading dye
6	clone K5	20 + 4x loading dye
7	clone K6	20 + 4x loading dye

250px

Conclusions:

• clones K1, K2 and K5 are ok

Further tasks:

• sequencing

Overnight cultures of pSB1C3+myc

Time: 2011-09-08, 18:30

Investigators: Jessica

Materials

• plates of pSB1C3+mycN and pSB1C3+mycC

• LB medium, Cm

Method:

• inoculation of 5 ml LB medium with Cm

• overnight at 37°C

Further tasks:

• test digest

• sequencing

Sidedirected mutagenesis of TEV protease and 14_3C protease

For better understanding of described experiment see also: http://141.89.201.101/iGEM/wiki2011/images/c/c8/UP_SG_Klonierungsschema_Protease.pptx

Investigators: Sascha, Paul, Sebastian

Aim: Sidedirected mutagenesis of TEV and 14_3C protease

Materials:

• TEV protease vector (P9 by Gunther Stier)

• 14_3C protease vector (P10 by Gunther Stier)

• different Primers

• Phusion polymerase kit by NEB

Methode:

PCR

• Template: 1 µL (pJC354 <10ng)

• Nucleotides: 1 µL of 10 mM ready to use dNTP mix
  

• 10 µL 5 x Phusion HF Buffer
  

• 2,5 µL primers = 25 pmol absolute (2,5 µL of each primer, see list below)
  

• 32,5 µL of pure water
  

• 0,5 µL Phusion HF polymerase
  

Program:

• Denat: 30 sec 98°C

• 30x

Denat: 10 sec 98°C

Anneal: 15 sec 69°C (with gradient of +/- 4°C)

Extend: 15 sec 72°C

• Final Extend: 10min 72°C

Used Primers:

Fragment 1 of TEV protease (T1):

• f_TEV_AraFusion_NgoMIV

• r_TEV_ACCAGC

Fragment 2 of TEV protease (T2):

• f_TEV_ACCAGC

• r_TEV_iGEM_BamHI

Fragment 1 of 14_3C protease (P1):

• f_14_3C_AraFusion_NgoMIV

• r_14_3C_ACCAGC

Fragment 2 of 14_3C protease (P2):

• f_14_3C_ACCAGC

• r_14_3C_tm_Xba208_A-T

Fragment 3 of 14_3C protease (P3):

• f_14_3C_tm_Xba208_A-T

• r_14_3C_tm_Xba280_A-T

Fragment 4 of 14_3C protease (P4):

• f_14_3C_tm_Xba280_A-T

• r_14_3C_iGEM_BamHI

Further Tasks:

PCR purification, analytical Gel to check sizes of the amplified fragments, assembly PCR of Fragment

Analytical gelelctrophoresis and PCR clean up of sidedirected mutated fragments T1, T2, P1, P2, P3 and P4 http://syntbio.net/iGEM/wiki2011/index.php/Labjournal_september#Sidedirected_mutagenesis_of_TEV_protease_and_14_3C_protease

For better understanding of described experiment see also: http://141.89.201.101/iGEM/wiki2011/images/c/c8/UP_SG_Klonierungsschema_Protease.pptx

Investigators: Sascha, Paul, Sebastian

Aim: Check the size of amplified fragments and clean them up after PCR

Materials:

• PCR fragments P1 to P4 and T1 and T2

• Fermentas 6x loading dye

• 100bp ladder (Gene Ruler by Fermentas)

• Macherey-Nagel Nucleo Spin II Kit for clean up

Methode:

• Clean up after manual for PCR purification of the used Kit

• gel electophoresis (2% agarose gel with analytical gel pockets): 30 min at 110 V

Results:

The sizes of the fragments of the PCR products are matching the fragment sizes extracted with Geneious:

• T1

• • sizes in Geneious: 411 bp

• • estimated sizes in gel: 410 bp

• T2

• • sizes in Geneious: 389 bp

• • estimated sizes in gel: 400 bp

• P1

• • sizes in Geneious: 167 bp

• • estimated sizes in gel: 170 bp

• P2

• • sizes in Geneious: 96 bp

• • estimated sizes in gel: 100 bp

• P3

• • sizes in Geneious: 99 bp

• • estimated sizes in gel: 100 bp

• P4

• • sizes in Geneious: 311 bp

• • estimated sizes in gel 310 bp

File:UP AG 2011-09-08-0140 muta TEV-Pre.JPG

DNA-Fragments after sidedirected mutagenesis:

• TEV fragment 1 (T1) c=

• TEV fragment 2 (T2) c=

• 14_3C fragment 1 (P1) c=

• 14_3C fragment 2 (P2) c=

• 14_3C fragment 3 (P3) c=

• 14_3C fragment 4 (P4) c=

Output:

Fragments T1 and T2, P1-P4 with expected sizes.

Further Tasks:

Merge of PCR fragments P1 and P2 into P5, P3 and P4 into P6, T1 and T2 into TEV-protease.

Assembly PCR of mutated TEV protease and 14_3C protease fragments

For better understanding of described experiment see also: http://141.89.201.101/iGEM/wiki2011/images/c/c8/UP_SG_Klonierungsschema_Protease.pptx

Investigators: Sascha, Stefan, Sebastian

Aim: Assembly of mutated TEV and 14_3C protease fragments

Materials:

• TEV protease fragments T1 and T2 --> TEV protease (total mutated)

• 14_3C protease fragments P1 and P2 --> P5

• 14_3C protease fragments P3 and P4 --> P6

• different Primers

• Phusion polymerase kit by NEB

Methode:

PCR

• Template: 2 µL (1µl of each fragment after sizes were checked and fragments got cleaned up with PCR clean up kit from Macherey-Nagel (Nucleo Spin Kit))

• Nucleotides: 1 µL of 10 mM ready to use dNTP mix
  

• 10 µL 5 x Phusion HF Buffer
  

• 2,5 µL primers = 25 pmol absolute (2,5 µL of each primer, see list below)
  

• 31,5 µL of pure water
  

• 0,5 µL Phusion HF polymerase
  

Program:

• Denat: 30 sec 98°C

• 30x

Denat: 10 sec 98°C

Anneal: 15 sec 70°C (with gradient of +/- 2°C)

Extend: 20 sec 72°C

• Final Extend: 10min 72°C

Used Primers:

Assembly of fragment 1 and fragment 2 of TEV protease (T1+T2) --> mutated TEV protease:

• f_TEV_AraFusion_NgoMIV

• r_TEV_iGEM_BamHI

Assembly of fragment 1 and fragment 2 of 14_3C protease (P1+P2)--> mutated 14_3C portease fragment 5 (P5):

• f_14_3C_AraFusion_NgoMIV

• r_14_3C_tm_Xba208_A-T

Assembly of fragment 3 and fragment 4 of 14_3C protease (P3+P4)--> mutated 14_3C portease fragment 6 (P6):

• f_14_3C_tm_Xba208_A-T

• r_14_3C_iGEM_BamHI

Further Tasks:

PCR purification, analytical Gel to check sizes of the amplified fragments, assembly PCR of Fragment

Analytical gelelctrophoresis and PCR clean up of sidedirected mutated fragments TEV protease, P5 and P6 http://syntbio.net/iGEM/wiki2011/index.php/Labjournal_september#Assembly_PCR_of_mutated_TEV_protease_and_14_3C_protease_fragments

For better understanding of described experiment see also: http://141.89.201.101/iGEM/wiki2011/images/c/c8/UP_SG_Klonierungsschema_Protease.pptx

Investigators: Sascha, Paul, Sebastian

Aim: Check the size of amplified fragments and clean them up after PCR

Materials:

• PCR fragments P5, P6 and TEV protease

• Fermentas 6x loading dye

• 100bp ladder (Gene Ruler by Fermentas)

• Macherey-Nagel Nucleo Spin II Kit for clean up

Methode:

• Clean up after manual for PCR purification of the used Kit

• gel electophoresis (1.5 % agarose gel with analytical gel pockets): 30 min at 110 V

Results:

The sizes of the fragments of the PCR products are matching the fragment sizes extracted with Geneious:

• TEV

• • sizes in Geneious:

• • estimated sizes in gel

• P5

• • sizes in Geneious:

• • estimated sizes in gel

• P6

• • sizes in Geneious:

• • estimated sizes in gel

(Picture from GelDoc and sizes will be added as soon as possible)

DNA-Fragments after sidedirected mutagenesis:

• TEV protease c=

• 14_3C fragment 5 (P5) c=

• 14_3C fragment 6 (P6) c=

Further Tasks: Merge of PCR fragments P5 and P6 into 14_3C protease, digestion of TEV protease with NgoMIV and BamHI, Ligation of TEV protease with digested AraC induction system (NgoMIV and HindIII) and digested pJC354_ssTorA_XhoI_CS-TEV_NheI_BlaFL (HindIII and BamHI)

Preparing glycerol stocks of pUP089

Time: 2011-09-08

Investigators: Jessica

Materials:

• overnight cultures of pUP089

• glycerol

Method:

• 700 µl culture

• 300 µl glycerol

• stored in -80°C blue iGEM box

Output:

• pUP089 Aa, Jes, 08.09.11

• pUP089 Ab, Jes, 08.09.11

• pUP089 Ba, Jes, 08.09.11

• pUP089 Bb, Jes, 08.09.11

Ligation of mdnA-lib2 and mdnA-Age

Time: 2011-09-08

Investigators: Nicole

Purification of phages containing pPDV089

Investigators: Sandrina

Aim: control if geneIII-mdnA will be expressed on the phage

Method/Materials:

• after over night incubation:

• centrifuge precipitaed phages: 5000 x g/ 45 min

• discard supernatant

• centrifuge again, 5000 x g/ 5 min

• remove supernatant carefully

• take pellet in 1 ml TBS

• move in 1.5 ml Eppi

• centrifuge again: 17000 x g/10 min

• supernatant in a new Eppi with 200 µl PEG-NaCl (mix!)

• incubate on ice for 60 min

• centrifuge precipitated phages: 17.000 x g/ 10 min (4°C)

• resuspend pellet in 300 µl TBS

• centrifuge:17 000 x g/ 10 min (4°C)

• supernatant in a fresh eppi= purified phages

Further tasks:

• measure concentration of phages
  

Glycerol stocks of phages containing pPDV089

Investigators: Sandrina

Aim: control if geneIII-mdnA will be expressed on the phage

Method/Materials:

• 300 µl phages in TBS

• 750 µl 86 % glycerol

• store at -80°C

Further tasks:

• measure concentration of phages
  

Concentration of phages containing pPDV089 measured by nanodrop

Investigators: Sandrina

Aim: control if geneIII-mdnA will be expressed on the phage

Results:

• OD 269: 0,235

• OD 320: 0,010

• phages per ml:

Ph/ml = (Abs 269- Abs 320) * 6*10^16/ bp (phagemid)

• bp (phagemid) = 10945

Ph/ml= 1,23*10^12

Further tasks:

• measure concentration of phages by serial dilution
  

plasmid pereperation of pARWIII to control deletion of kanamycin gene, pSB1C3 containing mdnA, geneIII or mdnA/geneIII

Investigators: Sandrina, Sabine

Aim: control deletion of kanamycin gene in created pARWIII, control ligation of mdnA, geneIII and mdnA/geneIII into pSB1C3

Method/Materials:

plasmid preperation:

protocol 5.1 of the NucleoSpin Plasmid Kit

Further tasks:

test digestion

Antibody detection with blotted membranes

Investigators: Sandrina

Aim: control if geneIII-myc-mdnA is presented on the phages

Method/Materials:

• incubate blocked membrane for 1 h with primary antibody (anti -c-myc- antibody) in TBS with 5% milk powder

• wash 3x 10 min with TBS buffer

• incubate for 1 h with secondary antibody

• wash 3x 10 min with TBS buffer

• develop membranes with ECL- Kit
  

Results:

• signals were only seen at positive controls and one negative control

Further tasks:

90th Labday 2011-09-09

Preparation of samples for HPLC analysis

Time: 2011-09-09

Investigators: Jessica, Katharina

Material:

• pellets of pARW089 and pARW071 samples (from 2011-09-06)

• 100% methanol

• 5& methanol

• sterile water

• Sep-Pak Cartridges

• speed vac

Method:

• pellet was resuspended in 5 ml of sterile water

• the resuspended sample was sonicated for 5 min (3 sec on, 3 sec off)

• centrifugation for 10 min @ 11000 rpm

• supernatant was transfered to Sep-Pak Plus C18 Cartridges, that were equilibrated with 2 ml of 100% methanol

• cartridges were washed with 2 ml water

• samples were load

• cartridges were washed with 2 ml of 5% methanol

• samples were eluted with 2 ml of 100% methanol

• samples were put in a speedvac so that the methanol can evaporate

Counting of colonies on plates (pUP089+lib2)

Time: 2011-09-09,7:30

Investigators: Nadine, Nicole

Aim: count number of colonies on plates

Material:

• agar plate (kan): 10x pUP089+lib2, Nadja, 7.9.11, resistance: kan, from 2011-9-8, Nad/Nic
  

Results:

plate	# of colonies	picked colonies
1	61	I clone E and F
2	38	II clone C and D
3	87	III clone A and B
4	47	IV clone G and H
5	33	IV clone I and J
6	55
7	37
8	70
9	28
10	36
sum	492

• example plates

Conclusions:

• 492 colonies

• some colonies will be picked for test sequencing (clone A-J)

• repeat fillin, ligation and transformation for more mutants

Glycerol stocks of pSB1C3+mdnDE K1, K2 and K5

Time: 2011-09-09, 8:00

Investigators: Nadine

Sequencing of pSB1C3+mdnD/ABC

Time: 2011-09-09, 8:00

Investigators: Nicole

Transformation of XL1 with different constructs

Time: 2011-09-09,

Investigators: Nadine, Nicole

Materials:

• XL1-Blue

• ligation products (2011-9-8):

• • mdnA-lib2

• • mdnA-Age

• • mdnABCDE from pARW071

• antibiotics from stock:

• • chloramphenicol

• • kanamycin

• • ampiciline

• LB-Agar

Method:

• ligation products:

• • addition of 2 µl ligation reaction to cells (XL1-blue) in 1.5 ml Eppi,

• incubation 30 min on ice,

• heat shock 60 sec at 42°C,

• incubation 5 min on ice,

• addition of 750 µl LB medium,

• incubation at 37 °C shaking for 60 min,

• plating on LB medium with appropriate antibiotic (20 ml Agar and 20 µl antibiotic (see above) per plate)

• storage over night at 37°C (start: 15:00)

Further tasks:

• Picking clones for overnight culture (except for library 2)

• Producing glycerol stocks

• count colonies of pUP089_lib2 and collect them w/ LB

Output:

• Plates w/ XL1 blue:

• • 10 x pUP089+mdnA-lib2 (on kan plates) + control

• • 10 x pARWIII+mdnA-Age (on amp plates) + control

• • pSB1C3+mdnABCDE from pARW071 (on cm plates) + control

Survival-Test of one 14_3C clone( SG9-P10) and one Tev-clone (SG13-T6)

Investigators: Paul, Stefan, Sascha

Aim: Test whether TorA-detection device functions

• SG13-T6 contains two silent mutations and one single amino-acid exchange (N-->D)

• SG9-P10 contains different mutations:...

Plates:

30x, 15x for each clone

• 2x Cm (25 µg/ml)

• 2x Cm + 1mM IPTG

• 2x Cm + 1mM Ara

• 2x Cm + Amp (50 µg/ml)

• 2x Cm + Amp (200 µg/ml)

• 2x Cm + 1mM Ara + Amp (50 µg/ml)

• 2x Cm + 1mM Ara + Amp (100 µg/ml)

• 2x Cm + 1mM Ara + Amp (200 µg/ml)

• 2x Cm + 1mM Ara + Amp (400 µg/ml)

• 2x Cm + 1mM Ara + Amp (800 µg/ml)

• 2x Cm + 1mM Ara + 1mM IPTG + Amp (50 µg/ml)

• 2x Cm + 1mM Ara + 1mM IPTG + Amp (100 µg/ml)

• 2x Cm + 1mM Ara + 1mM IPTG + Amp (200 µg/ml)

• 2x Cm + 1mM Ara + 1mM IPTG + Amp (400 µg/ml)

• 2x Cm + 1mM Ara + 1mM IPTG + Amp (800 µg/ml)

Method:

• cells from glycerolstocks (-80°C) were inoculated in 5 ml LB-Medium (Cm = 25µg/ml). Then diluted and incubated until OD=0.002

• 100µl of cells with OD=0.002 were plated on prepared plates

Results:

Survival screening have to be repeated, because there is no way to compare numbers of clones on plates (dual induction of protease via AraC and ssTorA_CS_blaFL construct via IPTG) with number of clones on the right controll plates (singel induction with IPTG to check the export via TAT-pathway of beta lactamase.

No resistance against ampicillin without induction with IPTG. Dual-induction with arabinose and IPTG does not lead to a dramatical reduced number of clones.

Further Tasks:

• Repeat the survival test with singel induction via IPTG as comparison for the cell death based on reduced export of beta lactamase into the periplasm of E. coli

• repeat survival test at 37 °C for both protease to check influence of creation of inclusion body of TEV protease at 37°C and influence of export capacity of beta lactamase via TAT-pathway

Assembly PCR of mutated 14_3C protease

For better understanding of described experiment see also: http://141.89.201.101/iGEM/wiki2011/images/c/c8/UP_SG_Klonierungsschema_Protease.pptx

Investigators: Sascha, Stefan, Sebastian

Aim: Assembly of mutated 14_3C

Materials:

• 14_3C protease fragments P5 and P6 --> 14_3C protease (total mutated)

• Primer 1: f_14_3C_AraFusion_NgoMIV

• Primer 2: r_14_3C_iGEM_BamHI

• Phusion polymerase kit by NEB

Methode:

PCR

• Template: 2 µL (1µl of each fragment after sizes were checked and fragments got cleaned up with PCR clean up kit from Macherey-Nagel (Nucleo Spin Kit))

• Nucleotides: 1 µL of 10 mM ready to use dNTP mix
  

• 10 µL 5 x Phusion HF Buffer
  

• 2,5 µL primers = 25 pmol absolute (2,5 µL of each primer, see list below)
  

• 31,5 µL of pure water
  

• 0,5 µL Phusion HF polymerase
  

Program:

• Denat: 30 sec 98°C

• 30x

Denat: 10 sec 98°C

Anneal/extend: 30 sec 72°C

• Final Extend: 10min 72°C

Further Tasks:

PCR purification, analytical Gel to check sizes of the amplified fragments. Digest and ligation.

Digestion of NgoMIV_TEV-Protease_iGEM_BamHI, NgoMIV_14_3C-Protease_iGEM_BamHI, HindIII_iGEM_AraC_NgoMIV fragments and pUP189-pJC354-NheI-TEV-Xho_blaFL_GGH5 and pUP189-pJC354-NheI-143C-Xho_blaFL_GGH5 vector

For better understanding of described experiment see also: http://141.89.201.101/iGEM/wiki2011/images/c/c8/UP_SG_Klonierungsschema_Protease.pptx

Investigators:Paul, Sascha, Stefan

Materials:

NgoMIV_TEV-Protease_iGEM_BamHI: 66,2 ng/µl

NgoMIV_14_3C-Protease_iGEM_BamHI: 91,7ng/µl

HindIII_iGEM_AraC_NgoMIV: 48,3 ng/µl

pUP189-pJC354-NheI-TEV-Xho_blaFL_GGH5 vector (contains TEV cleavage site): 470 ng/µl

pUP189-pJC354-NheI-143C-Xho_blaFL_GGH5 vector (contains 14_3C cleavage site): 270,2 ng/µl

Digestion protocol:

1: NgoMIV_TEV-Protease_iGEM_BamHI:

• 10µl NgoMIV_iGEM_TEV-Protease_iGEM_BamHI

• 1µl NgoMIV

• 1µl BamHI-HF

• 5µl 10x buffer = NEB 4

• 0.5µl 100x BSA

• 32.5µl pure water

• =50µl

2: NgoMIV_14_3C-Protease_iGEM_BamHI:

• 10µl NgoMIV_14_3C-Protease_iGEM_BamHI

• 1µl NgoMIV

• 1µl BamHI-HF

• 5µl 10x buffer = NEB 4

• 0.5µl 100x BSA

• 32.5µl pure water

• =50µl

3: HindIII_iGEM_AraC_NgoMIV:

• 10µl HindIII_iGEM_AraC_NgoMIV

• 1µl HindIII

• 1µl NgoMIV

• 5µl 10x buffer = NEB4

• 0.5µl 100x BSA

• 32.5µl pure water

• =50µl

4: pUP189-pJC354-NheI-TEV-Xho_blaFL_GGH5:

• 4µl pUP189-pJC354-NheI-TEV-Xho_blaFL_GGH5

• 1µl HindIII

• 1µl BamHI-HF

• 5µl 10x buffer = NEB 4

• 0.5µl 100x BSA

• 39.5µl pure water

• =50µl

5: pUP189-pJC354-NheI-143C-Xho_blaFL_GGH5:

• 8µl pUP189-pJC354-NheI-143C-Xho_blaFL_GGH5

• 1µl HindIII

• 1µl BamHI-HF

• 5µl 10x buffer = NEB 4

• 0.5µl 100x BSA

• 37.5µl pure water

• =50µl

-->The reaction was allowed to proceed for 2h at 37°C!

Ligation of NgoMIV_iGEM_14_3C-Protease_iGEM_BamHI or NgoMIV_iGEM_TEV-Protease_iGEM_BamHI, HindIII_iGEM_AraC_NgoMIV into pJC354-NheI-143C-Xho_blaFL_GGH5 or pJC354-NheI-TEV-Xho_blaFL_GGH5 vector

For better understanding of described experiment see also: http://141.89.201.101/iGEM/wiki2011/images/c/c8/UP_SG_Klonierungsschema_Protease.pptx

Investigators:Sebastian, Sascha, Stefan

Aim:

• 1. Triple-ligation of NgoMIV_iGEM_14_3C-Protease_iGEM_BamHI (573bp), HindIII_iGEM_AraC_NgoMIV (1273bp) and pJC354-NheI-143C-Xho_blaFL_GGH5 vector (~4700bp) to create pUP_SG2_TorA_CS-14_3C_bla_AraC-14_3C
  

• 2.Triple-ligation of NgoMIV_iGEM_TEV_iGEM_BamHI (760bp), HindIII_iGEM_AraC_NgoMIV (1273bp) and pJC354-NheI-TEV-Xho_blaFL_GGH5 vector (~4700bp)to create pUP_SG1_TorA_CS-TEV_bla_AraC-TEV
  

Calculation of volumes to be used with: [http://www.gibthon.org/ligate.html ligation calculator] with 1:1 molar ratio

Materials:

14_3C:

1:

• 1 µL NgoMIV_iGEM_14_3C-Protease_iGEM_BamHI fragment

• 1 µL AraC fragment

• 6 µL pJC354-NheI-143C-Xho_blaFL_GGH5 vector

• 1 µL T4 liagtion buffer (Fermentas)

• 1 µL T4 ligase (Fermentas)

• =10µl

• 1 controls: As 1 but with water instead of fragment

TEV:

• 1 µL NgoMIV_iGEM_TEV-Protease_iGEM_BamHI fragment

• 1 µL AraC fragment

• 6 µL pJC354-NheI-TEV-Xho_blaFL_GGH5 vector

• 1 µL T4 liagtion buffer (Fermentas)

• 1 µL T4 ligase (Fermentas)

• =10µl

• 1 control: As 4 but with water instead of fragment

TEV backbone with 14_3C:

• 1 µL NgoMIV_iGEM_14_3C-Protease_iGEM_BamHI fragment

• 1 µL AraC fragment

• 6 µL pJC354-NheI-TEV-Xho_blaFL_GGH5 vector

• 1 µL T4 liagtion buffer (Fermentas)

• 1 µL T4 ligase (Fermentas)

• =10µl

14_3C backbone with TEV:

• 1 µL NgoMIV_iGEM_TEV-Protease_iGEM_BamHI fragment

• 1 µL AraC fragment

• 6 µL pJC354-NheI-143C-Xho_blaFL_GGH5 vector

• 1 µL T4 liagtion buffer (Fermentas)

• 1 µL T4 ligase (Fermentas)

• =10µl

Used method:

ligation at room temperatur for 1h

Further task: Transformation of XL1blue cells with ligation products

Miniprep from overnight cultures T I/II, 14_3C CS,PC, TEV-CS-1, P1

For better understanding of described experiment see also: http://141.89.201.101/iGEM/wiki2011/images/c/c8/UP_SG_Klonierungsschema_Protease.pptx

Investigators: Niels

Aim: isolate DNA & sequencing

Samples for miniprep

• T I

• T II

• 14_3C-CS

• TEV-CS

• P1

• PC
  

Materials

miniprep by using Kit :

• NucleoSpin Plasmid

Nanodrop measurement

Sample	concentration [ng/µl]	260/280	260/230
T I	164,6	1,87	2,04
T II	182,7	1,88	2,07
14_3C-cs	263,1	1,86	2,13
TEV-cs	181,8	1,87	2,07
PC	150,5	1,85	2,05
P1	357,6	1,86	2,19

futher task :

• sequencing

Sequencing of miniprep (09-09-2011) - T I/II, 14_3C CS,PC, TEV-CS-1, P1

For better understanding of described experiment see also: http://141.89.201.101/iGEM/wiki2011/images/c/c8/UP_SG_Klonierungsschema_Protease.pptx

Investigators: Niels, Stefan

Samples: miniprep from 09-09-11

• T I

• T II

• TEV-cs

• 14_3C-cs

• P1

• PC

• each:

• • 1400 ng DNA

• • ad 20 µl steril water

sequenced by GATC

Transformation of TEV and 14_3C

For better understanding of described experiment see also: http://141.89.201.101/iGEM/wiki2011/images/c/c8/UP_SG_Klonierungsschema_Protease.pptx

Investigators:Sebastian, Sascha, Stefan

Aim:

Materials:

Heat shock protocol

Used method:

picking clones

Test digest of pSB1C3+mycN/C

Time: 2011-09-09

Investigators: Jessica

Aim: prove of Insert (mdnAmycN/C)

Materials:

• pSB1C3+mycN clones J1-J5, Niels, 9.9.11

• pSB1C3+mycC clones J1-J5, Niels, 9.9.11

• AvaII, PstI-HF (NEB)

• Buffer 4 (NEB, 10x)

• water

Plan:

insert	plasmid size in bp	enzymes	buffer	Expected size in bp
mdnAmycN	2596	AvaII, Pst-HF	4	225, 942, 1439
mdnAmycC	2597	AvaII, Pst-HF	4	225, 942, 1440

Digestion protocol

• 2 µl DNA

• 0.5 µl AvaII

• 0.5 µl PstI-HF

• 2 µl 10x buffer

• 15 µl pure water

• total: 20µl

• 37°C overnight
  

Agarose gel

lane	Sample	Volume in µl
M	Gene Ruler DNA Ladder Mix (1:10)	12
1	mycN clone 1	20 + 4x loading dye
2	mycN clone 2	20 + 4x loading dye
3	mycN clone 3	20 + 4x loading dye
4	mycN clone 4	20 + 4x loading dye
5	mycN clone 5	20 + 4x loading dye
6	mycC clone 1	20 + 4x loading dye
7	mycC clone 2	20 + 4x loading dye
8	mycC clone 3	20 + 4x loading dye
9	mycC clone 4	20 + 4x loading dye
10	mycC clone 5	20 + 4x loading dye
M	Gene Ruler DNA Ladder Mix (1:10)	12

Conclusions:

• digestion did not work out in a satisfying way

Further tasks:

• new restriction enzyme digestion

• sequencing

Miniprep of overnight cultures + pSB1C3+mycN/mycC

Time: 2011-09-09, 14:30

Investigators: Niels

Samples for miniprep

• pSB1C3+mycN J1

• pSB1C3+mycN J2

• pSB1C3+mycN J3

• pSB1C3+mycN J4

• pSB1C3+mycN J5

• pSB1C3+mycC J1

• pSB1C3+mycC J2

• pSB1C3+mycC J3

• pSB1C3+mycC J4

• pSB1C3+mycC J5
  

Materials

miniprep by using Kit :

• NucleoSpin Plasmid

Nanodrop measurement

Sample	concentration [ng/µl]	260/280	260/230
mycN-1	189,7	1,87	2,46
mycN-2	95,3	1,86	1,58
mycN-3	132,0	1,85	2,57
mycN-4	130,6	1,84	2,17
mycN-5	152,8	1,86	2,46
mycC-1	143,0	1,86	2,57
mycC-2	92,5	1,88	2,11
mycC-3	105,3	1,84	1,78
mycC-4	109,5	1,84	1,98
mycC-5	124,5	1,84	2,15

futher task :

• sequencing

Sequencing of pSB1C3+mdnDE/mycN/mycC

Time: 2011-09-09, 15:30

Investigators: Nicole, Niels, Jessica

Samples: miniprep from 09-09-11

• pSB1C3+mdnDE K1 (primers: VR2, sf_mdne_1_n (# 9), sf_mdne_2_1 (# 10), sf_mdne_3_1 (# 11))

• pSB1C3+mdnDE K2

• pSB1C3+mdnDE K5

• pSB1C3+mycN J1

• pSB1C3+mycN J2

• pSB1C3+mycN J3

• pSB1C3+mycN J4

• pSB1C3+mycN J5

• pSB1C3+mycC J1

• pSB1C3+mycC J2

• pSB1C3+mycC J3

• pSB1C3+mycC J4

• pSB1C3+mycC J5
  

• each:

• • 1400 ng DNA

• • ad 20 µl steril water

sequenced by GATC

Picking clones for overnight cultures of Lib 2 for confirmation

Investigators:Katharina

Time: 2011-09-09

Materials:

• agar plates with Lib2 clones

• LB medium

• kanamycin

Method:

• picking 10 clones

• • Lib2 clone A-J

• incubate over night in 37°C shaker (200 rpm)

Output:

• 10 liquid cultures Lib2

Further tasks:

• miniprep

• sending for sequencing

Prepare liquid culture for mdnA foc2

Investigators:Katharina

Time: 2011-09-09

Materials:

• agar plates with Lib2 clones

• LB medium

• kanamycin

Method:

• using 5 ml of LB media (without antibiotic) for sluicing down the colonies from the first plate

• transferring the media to the next plate

• procedure for all 10 plates

• incubate over night in 37°C shaker (200 rpm)

Output:

• 1 liquid cultures mdnA foc2

Further tasks:

• miniprep

Picking clones for overnight cultures of pSB1C3+mdnABCDE with and without T7-promotor

Investigators:Katharina

Time: 2011-09-09

Materials:

• agar plates of pSB1C3+mndABCDE with T7 promotor and pSB1C3+mdnABCDE without T7-promotor

• LB medium

• chloramphenicol

Method:

• picking 4 clones for pSB1C3+mdnABCDE without T7-promotor

• picking 2 clones for pSB1C3+mdnABCDE with T7-promotor

• incubate over night @ 37°C (200 rpm)

Output:

• 4 liquid cultures of pSB1C3+mdnABCDE without T7-promotor

• 2 liquid cultures of pSB1C3+mndABCDE with T7

Further tasks:

• miniprep

• sending for sequencing

Preparing overnight cultures of expression backbone-clones

Investigators:Katharina

Time: 2011-09-09

Materials:

• agar plates of expression backbones

• • pSB1A3_YFP_Ara

• • pSB1A3_YFP_Lac

• • pSB1K3_CFP_Ara

• • pSB1K3_CFP_Lac

• • pSB1K3_YFP_Lac

• LB medium

• ampicillin, kanamycin

Method:

• picking 1 clone for each construct into 10 ml LB media+antibiotic

• incubation overnight @ 37°C (200 rpm)

Output:

• 5 liquid cultures of different expression backbones

PCR: mdnABCDE

Time: 2011-09-09

Investigators: Nadja

Materials

• vector: pARW071 (1); pARW089 (2*)

• Long Enzyme Mix (+MgCl2) Buffer Fermentas

• Long Enzyme Mix Polymerase Fermentas

• dNTPs (NEB)

• water

• Primer:

#	Primer
84	pf_mdnABCDE089_NotI_SpeI_30.08.
83	pf_mdnABCDE_T7_EcoRI_NotI
82	r_mdnABCDE_iGEM

Protocol:

1. General composition

• 2 µl vector: pARW071 (1); pARW089 (2*) (diluted 1:100)

• 1 µl dNTPs (10 mM)

• 3 µl Primer forward (10 µM)

• 3 µl Primer backward (10 µM)

• 5 µl buffer

• 0.3 µl Ploymerase

• 35,7 µl water

• • total volume: 50 µl

2. Composition

• pARW071 (1): 84, 82

• pARW089 (2*)-T7: 83, 82

• pARW089 (2*): 84. 82

3. PCR programs

• IGLONG2
  

Further task

• gel verification

• purification

test digestion to control if pSB1C3 is containing mdnA, geneIII or mdnA/geneIII

Investigators: Sandrina, Steffi

Aim:control ligation of mdnA, geneIII and mdnA/geneIII into pSB1C3

Method/Materials:

• 5 clones from pSB1C3 with mdnA (chloramphenicol), 5 clones from pSB1C3 with geneIII (chloramphenicol), 5 clones from pSB1C3 with mdnA/geneIII (chloramphenicol):

• 4 µl vector DNA (58- 120 ng/µl)

• 2 µl buffer 3

• 0,2 µl BSA

• 0.5 µl XbaI

• 0.5 µl PstI

12.8 µl water

incubate for 1 h at 37°C

Results:

Loading of gels

lane	Sample	Volume in µl	Expected insert size in bp
M	marker, DNA ladder mix Fermentas
1	free
2	mdnA-geneIII fusion gene in pSB1C3, clone 1	12	ca. 700
3	mdnA-geneIII fusion gene in pSB1C3, clone 2	12	ca. 700
4	mdnA-geneIII fusion gene in pSB1C3, clone 3	12	ca. 700
5	mdnA-geneIII fusion gene in pSB1C3, clone 4	12	ca. 700
6	mdnA-geneIII fusion gene in pSB1C3, clone 5	12	ca. 700
7	geneIII in pSB1C3, clone 1	10	ca. 500
8	geneIII in pSB1C3, clone 2	10	ca. 500
9	geneIII in pSB1C3, clone 3	10	ca. 500
10	geneIII in pSB1C3, clone 4	10	ca. 500
11	geneIII in pSB1C3, clone 5	10	ca. 500
12	mdnA in pSB1C3, clone 1	12	ca. 180
13	mdnA in pSB1C3, clone 2	12	ca. 180
14	mdnA in pSB1C3, clone 3	12	ca. 180
15	mdnA in pSB1C3, clone 4	12	ca. 180
16	mdnA in pSB1C3, clone 5	12	ca. 180

Further tasks:

sequencing of clones

Send clones 2-6 (geneIII-mdnA in pSB) for sequencing

Investigators: Sandrina, Steffi

Aim:control ligation of mdnA/geneIII into pSB1C3

Method/Materials:

• Primer: VR

• 20 µl with 70 ng/µl DNA

Further tasks:

check alignment

test digestion to control if kanamycin resistance gene is knocked out of pARWIII

Investigators: Sandrina, Steffi

Aim:control knock out of kanamycin on pARWIII

Method/Materials:

• 5 clones:

• 4 µl vector DNA (100- 300ng/µl)

• 2 µl buffer 3

• 0,2 µl BSA

• 0.5 µl NsiI

• 13.3 µl water

incubate for 1 h at 37°C

Results:

Loading of gels

lane	Sample	Volume in µl	Expected insert size in bp
M	marker, DNA ladder mix Fermentas
1	pARWIII, clone 1	10	no insert band
2	pARWIII, clone 2	10	no insert
3	pARWIII, clone 3	10	no insert
4	pARWIII, clone 4	10	no insert
5	| 10	no insert	-

Further tasks:

sequencing of clones

91th Labday 2011-09-10

miniprep of Lib2 clones for confirmation

Time: 2011-09-10,9:00

Investigators: Niels, Nadine, Katharina

Materials:

• 10 overnight cultures (Lib2 clone A-J)

• NucleoSpin® Plasmid (NoLid) (Macherey-Nagel)

• • Protocol for high-copy plasmids

• • elution with 50 µl H₂O

• measuring concentration with NanoDrop:

Sample	concentration in ng/µl
Lib2 clone A	886.7
Lib2 clone B	979.7
Lib2 clone C	823.5
Lib2 clone D	815.5
Lib2 clone E	824.8
Lib2 clone F	987.7
Lib2 clone G	682.4
Lib2 clone H	683.4
Lib2 clone I	5.3
Lib2 clone J	767.5

Further Tasks:

• sending for sequencing

miniprep of mdnA foc2

Time: 2011-09-10,9:00

Investigators: Niels, Nadine, Katharina

Materials:

• 1 overnight culture

• NucleoSpin® Plasmid (NoLid) (Macherey-Nagel)

• • Protocol for high-copy plasmids

• • elution with 50 µl H₂O

• measuring concentration with NanoDrop:

Sample	concentration in ng/µl
mdnA foc2	610.0

Further Tasks:

• give it to the screening group

miniprep of pSB1C3+mdnABCDE with and without T7 promotor

Time: 2011-09-10

Investigators: Niels, Nadine, Katharina

Materials:

• 6 overnight cultures

• NucleoSpin® Plasmid (NoLid) (Macherey-Nagel)

• • Protocol for high-copy plasmids

• • elution with 50 µl H₂O

• measuring concentration with NanoDrop:

Sample	concentration in ng/µl
mdnABCDE with T7-promotor clone 1	6.1
mdnABCDE with T7-promotor clone 2	2.7
mdnABCDE without T7-promotor clone 1	12.1
mdnABCDE without T7-promotor clone 2	6.7
mdnABCDE without T7-promotor clone 3	4.9
mdnABCDE without T7-promotor clone 4	2.9

Further Tasks:

• doing new overnight culture and miniprep

Counting of colonies on plates (pUP089+lib2)

Time: 2011-09-09,10:00

Investigators: Nadine, Katharina, Niels

Aim: count number of colonies on plates

Material:

• agar plate (kan): 10x pUP089+lib2, Nic/Nad, 8.9.11, resistance: kan, from 2011-9-9, Nad/Kat
  

Results:

plate	# of colonies
1	78
2	92
3	65
4	55
5	67
6	80
7	116
8	87
9	66
10	35
sum	741
control	11

Conclusions:

• 741 colonies

• wash colonies from plates

• o.n. cultures and miniprep

• combine w/ preps from 2011-9-10 (Kat)

New test digest of pSB1C3+mycN/C

Time: 2011-09-10

Investigators: Niels, Nadine, Katharina

Aim: prove of Insert (mdnAmycN/C)

Materials:

• pSB1C3+mycN clones J1-J5, Niels, 9.9.11

• pSB1C3+mycC clones J1-J5, Niels, 9.9.11

• ScaI, XbaI (NEB)

• Buffer 3 (NEB, 10x)

• BSA

• water

Plan:

insert	plasmid size in bp	enzymes	buffer	Expected size in bp
mdnAmycN	2596	ScaI, XbaI	3	1024, 1570
mdnAmycC	2597	ScaI, XbaI	3	1024, 1570

Digestion protocol

• 1 µl DNA

• 0.5 µl ScaI

• 0.5 µl XbaI

• 2 µl 10x buffer

• 0.3 µl BSA

• 15.7 µl pure water

• total: 20µl

• 37°C for 1h
  

Agarose gel

lane	Sample	Volume in µl
M	Gene Ruler DNA Ladder Mix (1:10)	12
1	mycN clone 1	20 + 4x loading dye
2	mycN clone 2	20 + 4x loading dye
3	mycN clone 3	20 + 4x loading dye
4	mycN clone 4	20 + 4x loading dye
5	mycN clone 5	20 + 4x loading dye
6	mycC clone 1	20 + 4x loading dye
7	mycC clone 2	20 + 4x loading dye
8	mycC clone 3	20 + 4x loading dye
9	mycC clone 4	20 + 4x loading dye
10	mycC clone 5	20 + 4x loading dye
M	Gene Ruler DNA Ladder Mix (1:10)	12

Conclusions:

• digestion did not work out in a satisfying way

Test Expression Backbones: pSB1K3_Ara/Lac_CFP

Time: 2011-09-10,11:00

Investigators: Nadine, Katharina, Niels

Aim:

prove of promotors (Lac/Ara)

Materials/Methods

• start cell grow in fresh media

time	time of grow [min]	OD600 Ara_CFP	OD600 Ara_CFP control	OD600 Lac_CFP	OD600 Lac_CFP control
11:00	0	0,147	0,147	0,121	0,130
11:30	30	0,156	0,160	0,132	0,139
12:00	60	0,182	0,183	0,149	0,152
12:30	90	0,212	0,203	0,172	0,174
13:00	120	0,260	0,257	0,210	0,207
13:30	150	0,323	0,331	0,259	0,260
14:00	180	0,406	0,416	0,326	0,370
14:30	210	ind.	ind.	0,370	0,403
15:00	150	ind.	ind.	0,446	0,466

• after inducing by IPTG/ARA - samples where measured for 2h every 15 min

• • result negativ

further task

• repeat experiment

Glycerol stocks: Expression Backbones

Time: 2011-09-10,11:00

Investigators: Nadine, Katharina, Niels

Material:

• 300 µl DNA of the following expression backbones

• • pSB1A3_YFP_Ara

• • pSB1A3_YFP_Lac

• • pSB1K3_CFP_Ara

• • pSB1K3_CFP_Lac

• • pSB1K3_YFP_Lac

• • mdnA Lib-foc2

• 700 µl 86 % glycerol

• store at -80°C

Oligo-Fillin for mdnA-library 2 (repetition) and for Phage-display-mdnA-library (carrying AgeI restriction site)

Investigators: Niels, Nadine, Katharina

Time: 2011-09-10

Aim: Production of libraries carrying mutated mdnA, one for phage-display (carrying ageI restriction site)

Material:

• oligos (25 mM, HPLC purified, ordered by Sigma Aldrich):

• • o_mdnA_library, 76

• • o_focused_library_AgeI, 85

• 10x Klenow buffer

• dNTPs (10 mM)

• H2O

Method:

• Reaction mix (total volume: 50 µl)

• • 3 µl fw-oligonucleotide (o_mdnA-library, 76)

• • 3 µl rev-oligonucleotide (o_focused_library_AgeI, 85)

• • 5 µl dNTPs

• • 5 µl Klenow buffer

• • 34 µl H2O

• Reaction conditions

• • Program: ORIGAMI1

• after finishing program ORIGAMI1:

• • addition of 1 µl Klenow-fragment

• • incubation 1 hour, 37°C

Results:

• hybridized oligos for production of mdnA-library

Output:

• AgeI-library, named AgeI

Further tasks:

• PCR purification

• restriction enzyme digestion using SfoI and AgeI, also of pARWIII

Restriction enzyme digestion of ParWIII and fill-in-reaction of Libary 2

Investigators: Katharina, Niels, Nadine, Steffi

Time: 2011-09-10, 20:30

Material:

digest of Fill-in-Reaction of Libary 2 (2011-09-10) :

• 22,5 µl DNA (fill-in-product

• 2,5 µl Age I

• 2,0 µl Sfo I

• 3µl Buffer 3

• • 37°C over night

further task :

• purification by Kit

digestion of ParWIII:

• 6,5 µl template (pARWIII)

• 3 µl Buffer 4

• 2.5 µl Age I

• 2.0 µl Sfo I

• 16 µl water

• • 37°C over night

further task :

• purification by gel

further tasks:

• ligation

• transformation

Restriction enzyme digestion of pSB1C3 for ligation with mycN/C

Investigators: Nadine, Katharina, Niels

Time: 2011-09-10, 20:00

Material:

purified PCR products (2011-09-03) :

• mycC: 4.2 ng/µl

• mycN: 15.2 ng/µl

digestion of pSB1C3:

• 6 µl DNA (235.0 ng/µl (2011-06-22))

• 3 µl Buffer 4

• 1.5 µl XbaI

• 2.0 µl PstI

• 0.5 µl BSA

• 11.5 µl water

• • 37°C over night

further tasks:

• purification

• • ligation

• • transformation

Restriction enzyme digestion of pSB1C3 for control

Investigators: Niels, Nadine, Katharina, Steffi

Time: 2011-09-10, 20:30

Material:

pSB1C3(2011-06-22) :

• concentration: 235,0 ng/µl

digestion of pSB1C3 with Bgl I:

• 2 µl DNA (235.0 ng/µl (2011-06-22))

• 1 µl Buffer 3

• 0.5 µl BglI

• 6.5 µl water

• • total volume 10 µl

• 37°C over night

• expected fragments[bp]:

• • 1169

• • 962

• • 314

• • 199

digestion of pSB1C3 with SacI, TaqI:

• 2 µl DNA (235.0 ng/µl (2011-06-22))

• 1 µl Buffer 4

• 0.5 µl SacI

• 0,5 µl TaqI

• 0,3 µl BSA

• 5,7 µl water

• • total volume 10 µl

• 37°C over night

• expected fragments [bg]:

• • 1446

• • 648

• • 548

lane	Sample	Volume in µl
M	Gene Ruler DNA Ladder Mix (1:10)	12
1	pSB1C3 (BglI)	10 + 2 loading dye
2	pSB1C3 (SacI und TaqI)	10 + 2 loading dye

Picking clones for overnight cultures of pSB1C3+mdnABCDE

Investigators:Katharina, Steffi, Niels, Nadine

Time: 2011-09-10 20:45

Materials:

• agar plates of pSB1C3+mndABCDE (2011-09-09)

• LB medium

• chloramphenicol

Method:

• picking 5 clones for pSB1C3+mdnABCDE (2011-09-09)

• incubate over night @ 37°C (200 rpm)

Output:

• 5 liquid cultures of pSB1C3+mdnABCDE without T7-promotor

Further tasks:

• miniprep

• sending for sequencing

Preparing overnight cultures of expression backbone-clones

Investigators:Katharina, Niels, Nadine, Steffi

Time: 2011-09-10 19:30

Materials/Method:

• liquid culture :

• • pSB1K3_CFP_Ara

• • pSB1K3_CFP_Lac

• 10 ml LB medium

• 10 µl kanamycin

Output:

• 2 liquid cultures of expression CFP backbones with different promotors

Transformation of mdnABCDE + pSB1C3

Time: 2011-09-10 21:30

Investigators:Niels, Nadine, Katharina, Steffi

Aim: Transformation of E. coli cells with mdnABCDE + pSB1C3 (with and without T7) - ligation

Materials:

• competent E. coli cells (XL1-Blue, 2011-08-29)

• ligation products: mdnABCDE + pSB1C3

Method:

• addition of 2 µl ligation reaction to cells (XL1-blue) in 1.5 ml Eppi,

• incubation 20 min on ice,

• heat shock 90 sec at 42°C,

• incubation 10 min on ice,

• addition of 750 µl LB medium,

• incubation at 37 °C shaking(750 rpm) for 60 min,

• centrifuge 30 min at 2000 x g

• discard the supernatant (till ~100µl media)

• plating on LB plate with appropriate antibiotic (Cm)

• storage over night at 37°C

Further tasks:

• Picking clones for overnight culture

• Producing glycerol stocks

Output:

to be continued....

Colony PCR of plated TEV and 14_3C containing colonies

Investigators: Stefan, Paul, Sebastian

Aim:

get dna for BioBricks

Used Primer:

Tev:

f_TEV_iGEM, r_TEV_iGEM_BamHI

14_3C:

f_14_3C_iGEM, r_14_3C_iGem:BamHI

Method:

• 2,5µl of each primer (10µM) = 5µl

• 5µl 10y polymerase buffer

• 2µl 25mM MgCl2

• 1µl dNTP mix

• 0,5µl Taq-Polymerase (GenAxxon)

• 36,5 µl H2O

=50µl

Colonies were picked with a 20µl tip, dipped into PCR batch, and then plunged into 1ml LB to grew cells from picked colony (from these 1ml batches overnight cultures will be inoculated in case of positive clones).

• 10 colonies were picked for TEV and 10 colonies for 14_3C protease (see entry from 01.09.2011) = 20 PCR batches.

PCR Program:

Initial denat = 3min 94°C

25x

denat: 2min 10sec 94°C

anneal: 2min 10sec 70°C

extend: 1min 72°C

final extend: 10min 72°C

• The PCR products were resolved on a 1% analytical agarose gel

Results:

no bands!

Further Tasks:

repeat it!

Digest of TEV, 14_3C, AraC and different Vectors (pJS354_ssTorA_XhoI_CS-TEV_NheI_blaFL, pJS354_ssTorA_XhoI_CS-TEV_NheI_blaFL and pSB1C3)

Investigator: Nadine, Sebastian

Material:

Enzymes: (all pruchased from NEB)

• HindIII

• BamHI

• NgoMIV

• XbaI

• PstI

DNA-Fragments:

• mutated TEV protease

• mutated 14_3C protease

• AraC

• pJS354_ssTorA_XhoI_CS-TEV_NheI_blaFL

• pJS354_ssTorA_XhoI_CS-TEV_NheI_blaFL

• pSB1C3

NEBuffer 4, 3 and 1

BSA from NEB

Method:

All digestion batches got a total volume of 10µl and were incubated for 2 h at 37°C:

• 1µl Buffer

• 1µl each Enzyme

• 0,1 µl BSA if required

• 7 or 6,9 µl DNA

Both protease (TEV and 14_3C) got digested with BamHI+NgoMIV or NgoMIV+ PstI, AraC was digested with NgoMIV+HindIII or NgoMIV+XbaI. Both vector which containing the cleavage site for the Protease got digested with HindIII+BamHI, the vector pSB1C3 was digested with XbaI and PstI.

Output:

Digested fragements:

• NgoMIV_TEV_BamHI

• NgoMIV_TEV_PstI

• NgoMIV_14_3C_BamHI

• NgoMIV_14_3C_PstI

• HindIII_AraC_NgoMIV

• XbaI_AraC_NgoMIV

• BamHI_ pJS354_ssTorA_XhoI_CS-TEV_NheI_blaFL_HindIII

• BamHI_ pJS354_ssTorA_XhoI_CS-14_3C_NheI_blaFL_HindIII

• PstI_pSB1C3_XbaI

Further Tasks:

Clean up of all fragments via agarose gel and extraction of digested fragment from the agarose gel, ligation of fragments and teransformation into E. coli XL1 blue.

Gelextraction of digested fragements, ligation and transfomration of ligation products into E. coli XL1 blue

Investigator:

Sebastian, Sascha, Stefan

Aim:

Getting digested fragments purified, to get lost of small DNA fragments, we don't need.

Materials:

Digested fragements:

• NgoMIV_TEV_BamHI

• NgoMIV_TEV_PstI

• NgoMIV_14_3C_BamHI

• NgoMIV_14_3C_PstI

• HindIII_AraC_NgoMIV

• XbaI_AraC_NgoMIV

• BamHI_ pJS354_ssTorA_XhoI_CS-TEV_NheI_blaFL_HindIII

• BamHI_ pJS354_ssTorA_XhoI_CS-14_3C_NheI_blaFL_HindIII

• PstI_pSB1C3_XbaI

Methode:

Seperate digestion batches over an 1% agarose gel, cut of the bands with the right size and perform a gel purification with Nucleo spin II kit from Macherey-Nagel.

Output.

Digested and purified fragments of:

• NgoMIV_TEV_BamHI

• NgoMIV_TEV_PstI

• NgoMIV_14_3C_BamHI

• NgoMIV_14_3C_PstI

• HindIII_AraC_NgoMIV

• XbaI_AraC_NgoMIV

• BamHI_ pJS354_ssTorA_XhoI_CS-TEV_NheI_blaFL_HindIII

• BamHI_ pJS354_ssTorA_XhoI_CS-14_3C_NheI_blaFL_HindIII

• PstI_pSB1C3_XbaI

Further tasks:

Ligation of fragments into digested vector and transformation of E. coli XL1 blue with ligation products. Picking clones, performing a colony PCR, overnight cultures from all positive clones and creation of an glycerol stock culture and miniprep of those positive plasmids for sequencing.

Ligation of digested fragments of TEV protease, 14_3C protease and different vectors

Investigators: Stefan, Sascha, Paul

Materials:

Fragments:

• NgoMIV_TEV_BamHI

• NgoMIV_TEV_PstI

• NgoMIV_14_3C_BamHI

• NgoMIV_14_3C_PstI

• HindIII_AraC_NgoMIV

• XbaI_AraC_NgoMIV

• BamHI_ pJS354_ssTorA_XhoI_CS-TEV_NheI_blaFL_HindIII

• BamHI_ pJS354_ssTorA_XhoI_CS-14_3C_NheI_blaFL_HindIII

• PstI_pSB1C3_XbaI

QuickLigase purchased by Fermentas

QuickLigase 10x Buffer purchased by Fermentas

Methodes:

Standard ligation protocol from Fermantas for QuickLigase Kit. Ratio between vector and both fragments (protease and AraC-induction system) where 3:1:1.

Reaction batches:

Every batch contains 1µl QuickLigase 10x Buffer, and 0,5 µl QuickLigase (both by Fermentas).

• Batch 1:

• • NgoMIV_TEV_BamHI

• • HindIII_AraC_NgoMIV

• • BamHI_ pJS354_ssTorA_XhoI_CS-TEV_NheI_blaFL_HindIII

• Batch 2:

• • NgoMIV_14_3C_BamHI

• • HindIII_AraC_NgoMIV

• • BamHI_ pJS354_ssTorA_XhoI_CS-14_3C_NheI_blaFL_HindIII

• Batch 3:

• • NgoMIV_TEV_PstI

• • XbaI_AraC_NgoMIV

• • PstI_pSB1C3_XbaI

• Batch 4:

• • NgoMIV_14_3C_PstI

• • XbaI_AraC_NgoMIV

• • PstI_pSB1C3_XbaI

Every batch was incubated for 10 min at 37°C. After 10 min, all batches where put on ice.

Output:

• 4 different ligation batches

Further tasks:

• Transformation of E. coli XL1 blue with ligation products, colony PCR to get to know, which clones are positive and for all positive clones: mini-prep of plasmid DNA, sequencing and preparation of glycerol stock clutures.

Transfromation of E. coli XL1 blue with ligation products

Investigators: Sascha, Paul, Sebastian

Aim:

transformation of E. coli XL1 blue with ligation batcheshttp://www.syntbio.net/iGEM/wiki2011/index.php/Labjournal_september#Ligation_of_digested_fragments_of_TEV_protease.2C_14_3C_protease_and_different_vectors

Materials:

• 4 different ligation batches http://www.syntbio.net/iGEM/wiki2011/index.php/Labjournal_september#Ligation_of_digested_fragments_of_TEV_protease.2C_14_3C_protease_and_different_vectors

• competent E. coli XL1 blue cells

Method:

Standard transformation protocol using heat shock.

Output:

4 different E. coli cultures transformed with different ligation batches.

Further tasks:

plating of the transformed E. coli cultures on LB agar plates containing chloramphenicol.

Plating of transformed E. coli XL1 blue

Investigators: Stefan, Paul, Sebastian

Aim:

getting single positive E. coli XL1 blue colonies transformed with the different ligation batches http://www.syntbio.net/iGEM/wiki2011/index.php/Labjournal_september#Ligation_of_digested_fragments_of_TEV_protease.2C_14_3C_protease_and_different_vectors

Method:

100 µl of the transformed cultures where plated out on lB agar plates containing chloramphenicol, the rest of the transformed cultures where centrifuge at 4000 g for 3 min. The supernatant was discarded and the pellet re suspended in 100 µl LB media, which was also plated out on a new LB agar plate containing chloramphenicol. All agar plates where incubated over night at 37°C.

Further tasks:

picking clones for colony PCR and mini prep of plasmid DNA, also establishing of glycerol stock cultures and preparing plasmids for sequencing.