Team:Potsdam Bioware/Labjournal/August part 2

Contents 1 66th Labday 2011-08-17 1.1 Planning mdnA modification (library) 2 67th Labday 2011-08-18 2.1 Ordering oligos for modified mdnA library 2.2 miniprep of pSB1T3 clones containing CFP and YFP, respectively 2.3 restriction enzyme digestion of 2.4 Digestion of pak bla KDIR 2.5 Repeated PCR of mdnA and gene III for phage display (strategy 2) 2.6 Digestion of pSB1C3 3 69th Labday 2011-08-19 3.1 Clean up of digestion products 3.2 miniprep of pSB1A3 and pSB1K3 clones containing either CFP or YFP and different promotors 3.3 Ligation of pSB1T3 backbones with Ara, Lac and constitutive promotors 3.4 Transformation of competent RV cells with Ligation products 3.5 Site directed mutagenesis of 14_3C protease to remove iGEM restriction sites from the protease and introduction of iGEM restriction sites 3.6 Digestion of PCR products geneIII and mdnA for cloning into pSB1C3 3.7 Digestion of PCR products geneIII and mdnA for cloning mdnA/geneIII into pSB1C3 and pARW089 3.8 Digestion of PCR products geneIII and mdnA for cloning into pARW089 (3 fragment ligation) 3.9 Digestion of PCR product mdnA for cloning into pARW089 3.10 Analysis of sequenced pPDV100 3.11 Agarose gel electrophoresis of digested mdnA and geneIII 3.12 Repeated PCR of mdnA 3.13 Site directed mutagenesis of TEV protease to remove iGEM restriction sites from the protease and introduction of iGEM restriction sites and assembly of produced TEV-fragments 3.14 Amplificarion of arabinose induction system (AraC) from pBAD_iGEMexpress plasmid, produces a 1273bp fragment 3.15 Digestion of complete mutated TEV and 14_3C fragments and 3x-ligation into TEV- or 14_3C-backbones with amplified and digested AraC fragment 4 70th Labday 2011-08-20 4.1 miniprep of pSB1A3 and pSB1K3 clones containing either CFP or YFP and different promotors 4.2 Gel extraction of different AraC for TEV approaches 4.3 Ligation and transformation of 14_3C with backbone 4.4 production of competent E. coli XL 1 blue 4.5 Repeated digestion of PCR product mdnA for cloning into pARW089 (3 fragment ligation) 4.6 Agarose gel electrophoresis of digested mdnA 4.7 Ligation of geneIII and mdnA into pSB1C3 4.8 Ligation of geneIII and mdnA to get a fusion gene 4.9 Ligation of geneIII and mdnA into pARW089 (3 fragment ligation) 4.10 Ligation of mdnA into pARW089 5 71th Labday 2011-08-21 5.1 Transformation of competent RV cells with ligation products of pSB1T3/pSB1C3 backbones with Ara, Lac and constitutive promotors 5.2 miniprep of pSB1T3 clones containing CFP and YFP, respectively 5.3 Digest of pJC354_ssTorA_NheI_CS-14_3C_XhoI_blaFL 5.4 Assembly PCR of different TEV mutagenesis fraction 6 72th Labday 2011-08-22 6.1 Elimination of ampicillin resistance in pARW089 6.2 New primer ordered for cloning of geneIII into pARW089 (without mdnA) 6.3 Digestion of mdnA/geneIII fusion gene for cloning into pARW089 (2 step ligation) 6.4 Digestion of mdnA/geneIII fusion gene for cloning into pSB1C3 (2 step ligation) 6.5 Ligation of mdnA/geneIII into pARW089 6.6 Ligation of mdnA/geneIII into pSB1C3 6.7 Transformation of generated vector constructs in E. coli 6.8 Digest of pSB1C3, pSB1T3 clones containing CFP and YFP, respectively as well as PCR products of promoters 7 73th Labday 2011-08-23 7.1 overnight culture of picked E. coli clones transformed with pPDV 7.2 PCR: BioBrick mdnABC, mdnC, mdnE, mdnDE, mdnBC 7.3 Digest of pSB1T3+YFPI/II vectors for ligation w/ promotors 7.4 Digest of amplified 14_3C protease (assembled fragments after sidedirected mutagenesis), amplified AraC (from pBAD_iGEM_express mVenus), plasmid pJC354_ssTorA_XhoI_CS-143C_NheI_blaFL and plasmid pJC354_ssTorA_XhoI_CS-TEV_NheI_blaFL 7.5 Agarose Gel of PCR products for BioBricks mdnABC, mdnBC, mdnC, mdnC, mdnD, mdnDE, mdnE and PCR purification 7.6 Test digest of pSB1A3+CFP/YFP and pSB1K3+CFP/YFP vectors w/ promotors 7.7 Agarose Gel of pSB1A3+CFP/YFP and pSB1K3+CFP/YFP 7.8 Agarose Gel of pARW089 Ava II A (3µl DNA)/ B (4µl DNA) (= pUP089) 7.9 Ligation of pSB1C3 and pSB1T3-YFPI/II with promoters and ligation of pARW089 AvaII 7.10 Over night culture of several pSB1A3+CFP/YFP and pSB1K3+CFP/YFP with promotor 7.11 sequencing of 7.12 sequencing of 7.13 over night culture 8 74th Labday 2011-08-24 8.1 Transformation w/ pUP089 A and B, BBa_K40304_Affibody_MiddleLinker+Ara/Const/Lac, pSB1T3+YFPI+Ara/Const/Lac and pSB1T3+YFPII+Ara/Const/Lac 8.2 Miniprep of E.coli overnight culture containing created pPDV089 8.3 Test digestion of purified plasmids pARW089 8.4 Test digestion of purified plasmids pSB1C3 8.5 Agarose gel electrophoresis of digested mdn fragments and pSB1C3, gel extraction and ligation 8.6 Digest of pSB1A3+CFP/YFP and pSB1K3+CFP/YFP vectors w/ promotors 8.7 Transformation w/ pUP089 A and B, BBa_K40304_Affibody_MiddleLinker+Ara/Const/Lac, pSB1T3+YFPI+Ara/Const/Lac and pSB1T3+YFPII+Ara/Const/Lac 8.8 Miniprep of o. n. culture of pSB1K3 constitutive and Ara as well as pSB1A3 constitutive and Ara 9 75th Labday 2011-08-25 9.1 check plates from transformation (expression backbones in XL1-blue, 2011-08-24) 9.2 check plates from transformation (pUP089 in XL1-blue, 2011-08-24) 9.3 Sequencing of pSB1K3 constitutive and Ara as well as pSB1A3 constitutive and Ara from 2011-08-24 9.4 Transformation of pSB1C3 with several mdn genes/ promotor and pSB1T3-YFP I/II with several promotor 9.5 PCR of complete mdn cluster for biobrick-construction 9.6 gel purification of AraC, TEV, 14_3C and TEv vector and 14_3C vector 9.7 ligation of AraC, TEV/14_3C with the TEV/14_3C vector 9.8 transformation of ligations 9.9 over night culture of pUP089 A/*B from 24.08.2011 9.10 PCR of geneIII and mdnA 9.11 Analytic gel electrophoresis to control PCR 9.12 prepare samples for sequencing (created pPDV089) 9.13 digestion of created pPDV089 for deletion of kanamycin gene 9.14 Digestion of PCR products geneIII and mdnA for cloning into pSB1C3 9.15 Agarose gel electrophoresis for purification of digested PCR products 10 76th Labday 2011-08-26 10.1 miniprep of pUP089 clones 10.2 PCR of complete mdn cluster for biobrick-construction 10.3 digest of PCR products from 25.08.2011 10.4 Repeated ligation of digested mdn fragments and pSB1C3 ( from 2011-08-24) 10.5 Agarose gel and Gel extraction of pUP089 and the three libraries as well as their ligation 10.6 over night cultures of XL1 blue pSB1C3+mdn-combination 10.7 Glycerol stocks of E. coli containing created pPDV089 10.8 Gel electrophoresis to control digestion of pPDV089 with NsiI 10.9 PCR of geneIII 10.10 Ligation go geneIII and mdnA into pSB1C3 10.11 Re-ligation of NsiI-digested pPDVs089 10.12 Ligation of geneIII into pARW089 10.13 Transformation of created vectors in E. coli 11 77th Labday 2011-08-27 11.1 Agarose gel of PCR prducts mdnABC and PCR purification 11.2 competent cells - E.coli XL1 blue & RF308 11.3 miniprep of several mdn clones and glycerole stocks 11.4 Digestion of PCR products of complete mdn-cluster (mdnABCDE) 11.5 Transformation of ligation: pUP089 + Lib-1/2, pSB1c3 + several mdn 11.6 Ligation of 3x pUP089 + Lib-2 11.7 Ligation of mdnABCDE89 + pSB1C3 11.8 Mini-Prep of the positiv clones from an overnight culture 11.9 test digest of pSB1C3+mdncombinations (several mdn clones/combinations) 11.10 Troubleshooting of transformed cells 12 78th Labday 2011-08-28 12.1 comparison of "new" and "old" competent cells 12.2 check plates from transformation (pSB1C3 w/ mdn combinations in XL1 blue, 2011-08-27) 12.3 Agarose gel of PCR prducts mdnABC and PCR purification 12.4 over night cultures of XL1 blue pSB1C3+mdn-combination 12.5 Production of TEV and 14_3C biobricks part 1 12.6 Transformation of pSB1C3+mdnABCDE89 and pUP089+library2 12.7 overnight culture of picked E. coli clones transformed with pPDV089 to control deletion of kanamycin gene 13 79th Labday 2011-08-29 13.1 check transformation from yesterday (pSB1C3+mdnABCDE89 and pUP089+mdn-Lib2) 13.2 miniprep of several mdn clones 13.3 test digest of pSB1C3+mdncombinations (several mdn clones/combinations) 13.4 competent cells - E.coli XL1 blue 13.5 check resistance of competent cells - E.coli XL1 blue 13.6 Oligo-Fillin for mdnA-Library (repetition) 13.7 sequencing of TEV and 14_3C clones 13.8 Restriction of pUP089 (vector) and Lib-2/loop(Insert/PCR) 13.9 PCR of complete mdn cluster for biobrick-construction 13.10 Alignment of sequenced pPDV089 and model of pPDV089 13.11 PCR of geneIII and mdnA 14 80th Labday 2011-08-30 14.1 Agarose gel of PCR of complete mdn cluster for biobrick-construction 14.2 Repeat PCR of complete mdn cluster for biobrick-construction 14.3 Gel extraction and purification of pUP089 (vector) and Lib-2/loop(Insert/PCR) 14.4 Dephosphorylation and Ligation of pUP089 (vector) and Lib-2/loop(Insert/PCR) 14.5 Transformation of pUP089+library2 14.6 sequencing of mdnC, mdnD and mdnE BioBrick 14.7 check plates - resistance of competent E.coli XL1 blue cells 14.8 Assembly PCR for TEV and 14_3C 14.9 Digestion of created pPDV089 for deletion of kanamycin gene 14.10 Digestion of PCR products geneIII and mdnA for cloning into pSB1C3 14.11 Digestion of PCR product geneIII and vector pARW089 14.12 Ligation of geneIII into pARW089 14.13 Re-ligation of NsiI-digested pPDVs089 14.14 Ligation of geneIII and mdnA into pSB1C3 14.15 Transformation of created vectors in E. coli 14.16 gel electrophoresis of TEV and 14_3C 15 81th Labday 2011-08-31 15.1 Ligation of geneIII into pARW089 15.2 Transformation of pARW089 containing only geneIII (no mdnA) in E.coli 15.3 overnight culture of picked E. coli clones transformed with pPDV089 to control deletion of kanamycin gene, pARW089 containing only geneIII (no mdnA), pSB1C3 containing mdnA, geneIII or mdnA/geneIII 15.4 over night cultures of pSB1C3+mdnABC/mdnDE 15.5 Assembly PCR for 14_3C, PCR of AraC and TEV

66th Labday 2011-08-17

Planning mdnA modification (library)

Investigators: Nicole

Time: 2011-08-17, 20:45

Aim: Planning mdnA library

Materials

• codon table

• Kristian's diversity sheet

• excel

Results:

Output:

Excel Table: Y:\Klonierung\mdna modification\mdna modification 03.xlsx

67th Labday 2011-08-18

Ordering oligos for modified mdnA library

Investigators: Nicole

miniprep of pSB1T3 clones containing CFP and YFP, respectively

Investigators: Nicole, Jessica, Steffi, Katharina

Materials

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

• elution in 30µl

• Check concentration with NanoDrop
  

Results:

• pSB1T3+YFP I clone b: 72.1 ng/µl

• pSB1T3+YFP I clone c: 53.5 ng/µl

• pSB1T3+YFP I clone a: 197.1 ng/µl

• pSB1T3+YFP II plate 1 clone b: 46.1 ng/µl

• pSB1T3+YFP II clone a: 62.6 ng/µl

• pSB1T3+YFP II plate 2 clone d: 25.6 ng/µl

• pSB1T3+YFP II plate 2 clone c: 25.6 ng/µl

• pSB1T3+YFP I clone d: 101.8 ng/µl

Further Tasks:

• restriction enzyme digestion

• ligation with promotors

• transformation

restriction enzyme digestion of

Investigators: Nicole, Jessica, Katharina

Materials

• DNA of pSB1T3 clones and different promotors (Ara, Lac, constitutive)

• restriction enzymes: EcoRI, XbaI

• NEB Buffer 4

• water

Method

• 0.5µl EcoRI

• 0.5µl XbaI (again 0.5µl were added after 1.5 h)

• 1000ng DNA

• water added to total volume of 30µl

• restriction enzyme digestion overnight at 37°C

Digestion of pak bla KDIR

Investigators: Sabine

Aim:

• cutting out of geneIII for using as PCR template

Reason:

• pak bla KDIR contains a part of a myc tag

• the overhang of the geneIII forward primer contains a complete myc tag for insertion of myc into the phage display vector

• this may lead to the low yield of geneIII target DNA after PCR using entire vector pak bla KDIR as template

Time: 2011-08-18,10:00-14:00

Materials/Methods:

• 5 µl pak bla KDIR (ca 4500 ng)

• 2 µl NEB 10x buffer 2

• 1 µl restriction enzyme AvaI

• 1 µ restriction enzyme HindIII

• 11 µl water

• 4 h at 37°C

Further tasks:

• gel electrophoresis for purification of geneIII

• perform PCR of geneIII

Repeated PCR of mdnA and gene III for phage display (strategy 2)

Investigator: Sabine

Time: 2011-08-18, 15:00-18:00

Aim:

• amplification of geneIII with NgoMIV and AatII and rfc 25 restriction sites (strategy 2)
  

Reaction Components:

• 1 µl / 6 ng DNA (cut out geneIII from pak bla KDIR)

• 0,25 µl OneTaq Polymerase

• 1 µl dNTPs

• 1 µl per primer (pf_geneIII_Xba_NgoMIV_myc and pr_geneIII_iGEM_AatII)

• 5 µl 5x PCR Buffer

• 30,75 µl DNase free water

• purification of PCR fragments with QIAquick Gel Extraction Kit (250)

Further tasks:

• digestion / ligation

Digestion of pSB1C3

Investigators: Sabine

Aim:

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

Time: 2011-08-18,17:30-18:00

Materials/Methods:

• 8 µl pSB1C3 (ca 2000 ng)

• 2 µl NEB 10x buffer 2

• 1 µl restriction enzyme XbaI

• 1 µ restriction enzyme PstI

• 0,2 µl BSA

• 7,8 µl water

• over night at 37°C

Further tasks:

• gel electrophoresis for purification

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

69th Labday 2011-08-19

Clean up of digestion products

Investigators:Katharina

Materials

• products of restriction enzyme digestion

• NucleoSpin Extract II Kit from Macherey-Nagel

miniprep of pSB1A3 and pSB1K3 clones containing either CFP or YFP and different promotors

Investigators: Nicole, Jessica, Katharina

Materials

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

• elution in 50µl

• Check concentration with NanoDrop
  

Results

• all concentrations were around 30 ng/µl so the preps were thrown away

• new overnight cultures have been prepared

Further Tasks:

• new miniprep

• confirmation of clones by restriction enzyme digestion

• sending for sequencing

Ligation of pSB1T3 backbones with Ara, Lac and constitutive promotors

Investigators: Nicole, Jessica, Katharina

Materials

• T4 DNA Ligase Buffer

• T4 Ligase Buffer

• different EcoRi and XbaI digested pSB1T3 backbones

• • pSB1T3+YFP I clone b

• • pSB1T3+YFP I clone a

• • pSB1T3+YFP II plate 1 clone b

• • pSB1T3+YFP II clone a

• • pSB1T3+YFP I clone d: 101.8 ng/µl

• EcoRI and XbaI digested pSB1C3

• EcoRI and XbaI digested inserts

• • Ara-Promotor

• • constitutive promotor

• • Lac-Promotor

• water

Method

• 1µl 10x T4 DNA Ligase Buffer

• 1µl T4 DNA Ligase

• 2µl backbone DNA

• 5µl insert DNA

• 1µl water

• control was prepared for the constructs by taking water instead of insert DNA

Transformation of competent RV cells with Ligation products

Investigators:Jessica, Katharina

Materials

• ligation products of:

• • pSB1T3+YFPI clone b + different promotors

• • pSB1T3+YFPI clone a + different promotors

• • pSB1T3+YFPII plate 1 clone b + different promotors

• • pSB1C3 + different promotors

• competent RV cells

Method

• transformation was done using the heatshock-protocol

• pSB1T3 clones were plated on LB+agar+Tet

• pSB1C3 clones were plated on LB+agar+Cm

• incubation over night at 37°C

Site directed mutagenesis of 14_3C protease to remove iGEM restriction sites from the protease and introduction of iGEM restriction sites

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:

• Removal of iGEM restriction sites from 14_3C protease, amplifying protease fragments with iGEM restriction sites
  

Materials:

• Plasmid: pGEX-3_14_3C

• Primers: (1) f_14_3C_ACCAGC, r_14_3C_iGEM_BamHI (2) r_14_3C_ACCAGC, f_14_3C_AraFusion_NgoMIV, (3) f_14_3C_tm_Xbal208_A-T, r_14_3C_tm_Xbal208_A-T (4)r_14_3C_iGEM_BamHI, f_14_3C_tm_Xbal280_A-T

Used method:

PCR

• Template: 1µl

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

• 5µl 10x Amplification buffer S

• 2µl 25mM MgCl2

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

• 34,5µl of pure water

• 0,5µl TaqPol

Program:

• Denat: 3min 94°C

• 5x:

Denat: 45sec 94°C

Anneal:45sec 53°C

Extend:45sec 72°C

• 25x:

Denat: 45sec 60°C

Anneal:45sec 60°C

Extend:45sec 72°C

• Final Extend: 10min 72°C

Result:

Resolving of PCR products on preparative 2% agarose gel, exsciccion of coresponding band and gel exctraction via Nucleospin gel extract II.

Expected Fragments:

• 14_3C_mut_fragI: 153 bp

• 14_3C_mut_fragII: 66 bp

• 14_3C_mut_fragIII: 72 bp

• 14_3C_mut_fragIV: 260 bp

Further going:

• Assembly PCR of purificated products to produce NgoMIV_iGEM_14_3C-Protease_iGEM_BamHI (mutated 14_3C-Fragment)

Digestion of PCR products geneIII and mdnA for cloning into pSB1C3

Investigator: Sabine

Aim: cloning of mdnA and geneIII into pSB1C3

Time: 2011-08-19, 10:00-13:00

Material/Method:

• 30 µl PCR product (990 ng mdnA or 3200 ng geneIII)

• 1 µl restriction enzyme XbaI

• 1 µl restriction enzyme PstI

• 0,5 µl BSA

• 4 µl NEB 10x buffer 2

• 3,5 µl water

• 1 h, 37°C

Further Tasks:

• gel electrophoresis for purification

• cloning into pSB1C3

Digestion of PCR products geneIII and mdnA for cloning mdnA/geneIII into pSB1C3 and pARW089

Investigator: Sabine

Aim: cloning of mdnA/geneIII fusion gene into pSB1C3 and pARW089 (2 step ligation)

Time: 2011-08-19, 10:00-13:00

Material/Method:

• 30 µl / 990 ng mdnA

• 1 µl restriction enzyme AgeI

• 4 µl NEB 10x buffer 1

• 5 µl water

• 1 h, 37°C

• 30 µl / 3200 ng geneIII

• 1 µl restriction enzyme NgoMIV

• 4 µl NEB 10x buffer 4

• 5 µl water

• 1 h, 37°C

Further Tasks:

• gel electrophoresis for purification

• ligation of mdnA and geneIII, than ligation into pSB1C3

Digestion of PCR products geneIII and mdnA for cloning into pARW089 (3 fragment ligation)

Investigator: Sabine

Aim: cloning of mdnA and geneIII into pARW089 (3 fragment ligation)

Time: 2011-08-19, 10:00-13:00

Material/Method:

• 30 µl / 990 ng mdnA

• 2 µl restriction enzyme NarI

• 1 µl restriction enzyme AatII

• 4 µl NEB 10x buffer 4

• 3 µl water

• 1 h, 37°C

• 30 µl / 3200 ng geneIII

• 1 µl restriction enzyme NgoMIV

• 1 µl restriction enzyme AatII

• 4 µl NEB 10x buffer 4

• 4 µl water

• 1 h, 37°C

Further Tasks:

• gel electrophoresis for purification

• ligation into pARW089

Digestion of PCR product mdnA for cloning into pARW089

Investigator: Sabine

Aim: cloning of mdnA into pARW089

Time: 2011-08-19, 10:00-13:00

Material/Method:

• 30 µl / 990 ng mdnA

• 2 µl restriction enzyme NarI

• 1 µl restriction enzyme AatII

• 4 µl NEB 10x buffer 4

• 3 µl water

• 1 h, 37°C

Further Tasks:

• gel electrophoresis for purification

• ligation into pARW089, than digestion with AgeI and NgoMIV and ligation of geneIII

Analysis of sequenced pPDV100

Investigator: Sabine

Aim: control of created pPDV100

Time: 2011-08-19, 14:00-14:15

Result:

• created vector contains no mdnA

Further Tasks:

• this strategy will not be further persued

Agarose gel electrophoresis of digested mdnA and geneIII

Investigator: Sabine

Aim: purification of digested DNA fragments

Time: 2011-08-19, 15:00-17:00

Material/Method:

• digested geneIII, mdnA, pSB1C3

• 1% agarose gel, loading dye, DNA ladder mix (Fermentas)

• 100 V

Results:

• sample mdnA (NarI+AatII) has been lost (ran under the gel)

• concentration pSB1C3 (Xba+Pst): 17 ng/µl

• concentration mdnA (AgeI): 3,8 ng/µl

• concentration mdnA (XbaI+PstI): 5,5 ng/µl

• concentration geneIII (NgoMIV): 15,5 ng/µl

• concentration geneIII (XbaI+PstI): 12,2 ng/µl

• concentration geneIII (NgoMIV+AatII): 12,8 ng/µ
  

Further Tasks:

• repeat PCR and digestion of mdnA (NarI+AatII)

• ligations

Repeated PCR of mdnA

Investigator: Sabine

Time: 2011-08-19,17:00-19:00

Aim:

• amplification of mdnA with NarI and AgeI restriction sites
  

Primer:

• primer: pf_mdnA_iGEM_EheI and pr_mdnA_iGEM_AatII
  

Reaction Components:

• 2 µl Vector pARW089 (16 ng)

• 0,25 µl Taq Polymerase S (BioScience)

• 1 µl dNTPs

• 1 µl per primer

• 5 µl 10x PCR Buffer S

• 39,75 µl water

Further tasks:

• purification

• digestion

Site directed mutagenesis of TEV protease to remove iGEM restriction sites from the protease and introduction of iGEM restriction sites and assembly of produced TEV-fragments

Investigators: Sascha, Paul

• Mutagenesis was performed as described before in the wiki.

• 3 reaction batches

Results:

Expected fragments (360bp and 400bp) were excissed and extracted from the gel using Nucleospin Extract Kit

• Assembly PCR of TEV fragments I and II was performed as described before in the wiki

The expected mutated complete TEV fragment was PCR purificated using nucleospin extract KIT

Amplificarion of arabinose induction system (AraC) from pBAD_iGEMexpress plasmid, produces a 1273bp fragment

Investigators: Sascha, Paul, Sebastian

Materials:

Plasmid: pBAD_iGEMexpress (Nr.4) Primers: f_AraC_HindIII_iGEM , r_AraC_NgoMIV

Used method: PCR

Template: 1µl = 7,2 ng

Nucleotides: 1 µl of 10mM ready to use dNTP mix 5µl 10x

Amplification buffer S: 5µl 25mM MgCl2 2,5µl

primers = 25pmol absolute (2,5µl of each primer = 5µl per tube) 32,5µl of pure water 0,5µl TaqPol

Program: iGEM002

Denat: 3min 94°C

5x:

Denat: 60sec 94°C

Anneal:60sec 53°C

Extend:60sec 72°C

25x:

Denat: 60sec 60°C

Anneal:60sec 60°C

Extend:60sec 72°C

Final Extend: 10min 72°C

• PCR purification using NucleoSpin Extract KIT and digestion of AraC fragment (1273bp) witth NgoMIV and HindIII

• Resolving of digested AraC fragment on 1.5% preparative agarose Gel

• The corresponding band (1273bp) was excissed and extracted from the gel using Nucleospin Extract KIT.

Expected Fragments: HindIII_iGEM_AraC_NgoMIV 1273bp

Digestion of complete mutated TEV and 14_3C fragments and 3x-ligation into TEV- or 14_3C-backbones with amplified and digested AraC fragment

Investigators: Paul, Stefan

Materials:

(1)NgoMIV_iGEM_TEV-Protease_iGEM_BamHI

(2)NgoMIV_iGEM_14_3C-Protease_iGEM_BamHI

(3)HindIII_iGEM_AraC_NgoMIV (digested)

• digestion of proteases (1) and (2) with NgoMIV and BamHI as described before in the wiki with NgoMIV and BamHI

• resolving of digested fragments on preparative agarose gel and excission of corresponding bands. Extraction of DNA from the gel was performed using NucleoSping ExtractII KIT.

Ligation of digested proteases into digested the pJC354-14_3C/TEV vectors including the AraC fragment (3)

concentrations for ligations were calculated using gibthon ligation calculator: [http://www.gibthon.org/ligate.html CALC]

• ligations were allowed to proceed for 1h at room temperature and were immediately transformed into competent XL1-blue cells using standard transformation protocol

70th Labday 2011-08-20

miniprep of pSB1A3 and pSB1K3 clones containing either CFP or YFP and different promotors

Investigator: Jessica, Katharina

Materials

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

• elution in 50µl

• Check concentration with NanoDrop
  

Results

• concentrations:

• • pSB1A3-CFP +Ara (clone A) 12: 342.1 ng/µl

• • pSB1A3-CFP +Ara (clone B) 7: 252.5 ng/µl

• • pSB1A3-CFP +Ara (clone C) 6: 356.9 ng/µl

• • pSB1A3-YFP +Ara (clone A) 1: 339.2 ng/µl

• • pSB1A3-YFP +Ara (clone B) 5: 379.0 ng/µl

• • pSB1A3-YFP +Ara (clone C) 14: 221.4 ng/µl

• • pSB1A3-CFP +Lac (clone A) 15: 319.3 ng/µl

• • pSB1A3-CFP +Lac (clone B) 34: 105.5 ng/µl

• • pSB1A3-CFP +Lac (clone C) 33: 135.7 ng/µl

• • pSB1A3-YFP +Lac (clone A) 16: 110.6 ng/µl

• • pSB1A3-YFP +Lac (clone B) 4: 340.4 ng/µl

• • pSB1A3-YFP +Lac (clone C) 13: 290.6 ng/µl

• • pSB1A3-CFP + const (clone A) 11: 312.2 ng/µl

• • pSB1A3-CFP + const (clone B) 9: 218.0 ng/µl

• • pSB1A3-CFP + const (clone C) 10: 201.7 ng/µl

• • pSB1A3-YFP + const (clone C) 28: 238.0 ng/µl

• • pSB1K3-CFP +Ara (clone A) 25: 175.2 ng/µl

• • pSB1K3-CFP +Ara (clone B) 31: 190.2 ng/µl

• • pSB1K3-CFP +Ara (clone C) 24: 193.8 ng/µl

• • pSB1K3-YFP +Ara (clone A) 22: 119.6 ng/µl

• • pSB1K3-YFP +Ara (clone B) 19: 149.4 ng/µl

• • pSB1K3-YFP +Ara (clone C) 30: 135.1 ng/µl

• • pSB1K3-CFP +Lac (clone A) 20: 138.4 ng/µl

• • pSB1K3-CFP +Lac (clone B) 21: 58.3 ng/µl

• • pSB1K3-CFP +Lac (clone C) 23: 122.0 ng/µl

• • pSB1K3-YFP +Lac (clone A) 29: 69.3 ng/µl

• • pSB1K3-YFP +Lac (clone B) 17: 153.1 ng/µl

• • pSB1K3-YFP +Lac (clone C) 18: 90.9 ng/µl

• • pSB1K3-CFP + const (clone A) 26: 89.8 ng/µl

• • pSB1K3-CFP +const (clone B) 8: 252.5 ng/µl

• • pSB1K3-CFP +const (clone C) 2: 256.6 ng/µl

• • pSB1K3-YFP + const (clone A) 27: 55.7 ng/µl

• • pSB1K3-YFP +const (clone B) 3: 231.9 ng/µl

• • pSB1K3-YFP + const (clone C) 32: 174.9 ng/µl
    

• • pARW089 35: 333.8 ng/µl

• • pARW089 36: 326.4 ng/µl

Further Tasks:

• confirmation of clones by restriction enzyme digestion

• sending for sequencing

Gel extraction of different AraC for TEV approaches

Investigator: Stefan

Aim:

• purification of DNA
  

Materials:

• NucleoSpin Kit (Machery Nagel)
  

Method:

• DNA extraction from agarose gels protocol of NucleoSpin Kit
  

Results:

BILD von Sabine geschickt, von mir eingefügt

Further tasks:

Ligation with TEV and 14_3C and appropriate backbone

Ligation and transformation of 14_3C with backbone

Investigator: Paul, Stefan

Aim:

• liagte and transform 14_3C with AraC and backbone
  

Materials:

Transformation Protocol Using Heat Shock

1) Take chemically competent E. coli cells from –80°C freezer.

a. Use XL1-blue cells for all cloning and DNA-related tasks, BL21 cells for protein/peptide expression.

2) Turn on water bath or heat block to 42°C.

3) Competent cells should be in a 1.5 ml tube. For transforming a DNA construct, use 60 ul of competent cells. .

4) Keep tubes on ice.

5) Add DNA solution into the E.coli cell suspension, mix by flicking the tube. Incubate on ice for 15-30 min.

Note: 2 µL from a T4 DNA ligase reaction are usually sufficient

6) Put tubes into heat block at 42°C for 45 seconds.

7) Put tubes back on ice for 2 minutes to reduce damage to the E. coli cells.

8) Add 750 µL of LB or DYT (with no antibiotic!). Incubate tubes for 1 hour at 37°C and 750 rpm.

9) Spread 100 ul of the resulting culture on LB agar plates (with appropriate antibiotic added!).

Note: In case of "tricky" ligations which yield few colonies, spin down the cell suspension for 3 min at 6000 rcf (since you can't spread 800 µL), discard most of the supernatant, resuspend the bacterial pellet by pipetting and use this for spreading.

10) Grow overnight at 37 °C.

11) Pick colonies about 12-16 hours later.

Method:

• 
  

Results:

check clones via colony PCR for correct insert

Further tasks:

sequencing postive clones

production of competent E. coli XL 1 blue

Investigator: Stefan

Aim:

• competent E. coli XL 1 blue
  

Materials:

• CaCl2

• E. coli XL1 blue

Method:

Work always sterile and cold and speedy!

• All volumes deal with the common cell line!

• The cooling-centrifuge is in the tool shed , cool down to 4°C early enough , close the lid correctly!

• Prepare early enough min. 100 Eppis (1,5µl) (per cellline) and cool down to -80°C before using

• Use Milipore-filter for sterile CaCl₂ , keep cool!

• prepare 15ml LB-Medium (or DYT) with the specific antibiotic (XL1-blue? Tet, BL21 ? none!), inoculate and incubate over night

• prepare 200ml LB-Medium (or DYT) with the specific antibiotic, inoculate with 2ml of the over-night-culture. Nurture the culture until OD600 at 0,35 (0,2-0,5) (if the OD is too high, the cell won’t be competent)

• keep cell suspension in sterile falcons (50ml) 20 min on ice, then centrifuge for 20min; 4°C; 2500g

• discard supernatant, carefully resuspend on ice with 10ml cold CaCl₂-solution (put a little of the 10ml solution in every falcon before!), pool every resuspended aliquot of one cell line and add 40ml CaCl₂-solution (total volume 50ml). Keep 30 min on ice, then centrifuge for 20min; 4°C; 2500g

• discard supernatant, carefully resuspend pellet in 5,5ml CaCl₂(80mM)/Glycerol (4:1), aliquot in Eppis á 60µl and store immediately at - 80 °C

Repeated digestion of PCR product mdnA for cloning into pARW089 (3 fragment ligation)

Investigator: Sabine

Aim: cloning of mdnA and geneIII into pARW089 (3 fragment ligation)

Time: 2011-08-20, 10:00-11:30

Material/Method:

• 30 µl / 920 ng mdnA

• 2 µl restriction enzyme NarI

• 1 µl restriction enzyme AatII

• 4 µl NEB 10x buffer 4

• 3 µl water

• 1 h, 37°C

Further Tasks:

• gel electrophoresis for purification

• ligation into pARW089

Agarose gel electrophoresis of digested mdnA

Investigator: Sabine

Aim: purification of digested DNA fragments

Time: 2011-08-20, 11:30-12:30

Material/Method:

• digested mdnA

• 1% agarose gel, loading dye, DNA ladder mix (Fermentas)

• 100 V

Further Tasks:

• ligations

Ligation of geneIII and mdnA into pSB1C3

Investigator: Sabine

Time: 2011-08-20, 13:00-16:00

Material/Method:

• 1 µl mdnA/XbaI+PstI (5,5 ng/µl)

• 10 µl pSB1C3/XbaI+PstI(17 ng/µl)

• 2 µl 10x T4 ligase buffer

• 1 µl T4 ligase

• 6 µl water

• 1 h, room temperature

• 10 µl geneIII/XbaI+PstI (12,2 ng/µl)

• 1 µl pSB1C3/XbaI+PstI (17 ng/µl)

• 2 µl 10x T4 ligase buffer

• 1 µl T4 ligase

• 6 µl water

• 1 h, room temperature

Further Tasks:

• transformation

Ligation of geneIII and mdnA to get a fusion gene

Investigator: Sabine

Time: 2011-08-20, 13:00-16:00

Material/Method:

• 10 µl mdnA/AgeI (3,8 ng/µl)

• 5,6 µl geneIII/NgoMIV (15,5 ng/µl)

• 2 µl 10x T4 ligase buffer

• 1 µl T4 ligase

• 1,4 µl water

• 1 h, room temperature

Further Tasks:

• digestion with NarI and AatII, ligation into pARW089

• digestion with XbaI and PstI, ligation into pSB1C3

Ligation of geneIII and mdnA into pARW089 (3 fragment ligation)

Investigator: Sabine

Time: 2011-08-20, 13:00-16:00

Material/Method:

• 10 µl pARW089/NarI+AatII (60 ng/µl)

• 1 µl geneIII/NgoMIV+AatII (79 ng/µl)

• 1,5 µl mdnA/NarI+AgeI (24 ng/µl)

• 2 µl 10x T4 ligase buffer

• 1 µl T4 ligase

• 4,5 µl water

• 1 h, room temperature

Further Tasks:

• transformation

Ligation of mdnA into pARW089

Investigator: Sabine

Time: 2011-08-20, 13:00-16:00

Material/Method:

• 10 µl pARW089/NarI+AatII (60 ng/µl)

• 1 µl mdnA/NarI+AatII (47 ng/µl)

• 2 µl 10x T4 ligase buffer

• 1 µl T4 ligase

• 6 µl water

• 1 h, room temperature

Further Tasks:

• digestion of ligation construct with AgeI and AatII, ligation of geneIII/NgoMIV+AatII (2 step ligation)

71th Labday 2011-08-21

Transformation of competent RV cells with ligation products of pSB1T3/pSB1C3 backbones with Ara, Lac and constitutive promotors

Investigator: Katharina

Idea

• because of unclear labeling the RV cells used on 2011-08-19 could have been XL1 blue, which already have Tet resistance

Material

• pSB1T3 clones

• • pSB1T3+YFPI clone b (labeled with "1")

• • pSB1T3+YFPI clone a (labeled with "3")

• • pSB1T3+YFPII plate I clone b (labeled with "4")

• pSB1C3

• competent RV cells

Method

• transformation was done using the heatshock-protocol

• pSB1T3 clones were plated on LB+agar+Tet

• pSB1C3 clones were plated on LB+agar+Cm

• incubation over night at 37°C

Further Tasks

• pick clones for liquid culture

• miniprep

• restriction enzyme digestion for confirmation

• send for sequencing

miniprep of pSB1T3 clones containing CFP and YFP, respectively

Investigators: Katharina

Materials

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

• elution in 50µl

• Check concentration with NanoDrop
  

Results:

• pSB1T3+YFP I clone b: 690.6 ng/µl

• pSB1T3+YFP I clone c: 576.3 ng/µl

• pSB1T3+YFP I clone a: 646.9 ng/µl

• pSB1T3+YFP II plate 1 clone b: 599.8 ng/µl

• pSB1T3+YFP II clone a: 562.8 ng/µl

• pSB1T3+YFP II plate 2 clone d: 569.1 ng/µl

• pSB1T3+YFP II plate 2 clone c: 415.0 ng/µl

• pSB1T3+YFP I clone d: 559.9 ng/µl

Digest of pJC354_ssTorA_NheI_CS-14_3C_XhoI_blaFL

Investigator: Sascha, Sebastian

Aim:

• Digestion of vector pJC354_ssTorA_NheI_CS-14_3C_XhoI_blaFL for ligation with AraC and 14_3C protease
  

Materials:

• 4 µl of 3 different fractions of pJC354_ssTorA_NheI_CS-14_3C_XhoI_blaFL (approx. 1,2-1,5 µg DNA)
  

• Restriction enzymes BamHI HF and HindIII (purchased form NEB)
  

• Buffer 4 (purchased from NEB)
  

Method:

• Standard digestion protocol for plasmid DNA
  

Results:

• 3 different digested vector fractions of pJC354_ssTorA_NheI_CS-14_3C_XhoI_blaFL for ligation with AraC and 14_3C protease
  

Further tasks:

• purification via gel purification (Kit from Macherey-Nagel)
  

Output:

• pJC354_ssTorA_NheI_CS-14_3C_XhoI_blaFL vector fraction 1, c= ng/ml
  

• pJC354_ssTorA_NheI_CS-14_3C_XhoI_blaFL vector fraction 2, c= ng/ml
  

• pJC354_ssTorA_NheI_CS-14_3C_XhoI_blaFL vector fraction 3, c= ng/ml
  

Assembly PCR of different TEV mutagenesis fraction

Investigator: Sascha, Sebastian

Aim:

• Assembly of the different fractions of mutated TEV-fragments
  

Materials:

• TEV mutagenesis fragment I (fraction I, II, III, IV)
  

• TEV mutagenesis fragment II (fraction I, II, III, IV)
  

• Primer 1: f_TEV_AraFusion (43)
  

• Primer 2: r_TEV_iGEM_BamHI (46)
  

• Taq-Polymerase (purchased from Genaxxon)
  

• 10x polymerase buffer (purchased from Genaxxon)
  

• 10 mM (each) dNTPs (purchased from Genaxxon)
  

• double destilled water
  

• 25 mM MgCl2
  

Method:

PCR

• Template 1: 1µl TEV mutagenesis fragment I (fraction I-IV)
  

• Template 2: 1µl TEV mutagenesis fragment II (fraction I-IV)
  

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

• 5µl 10x Amplification buffer S
  

• 2µl 25mM MgCl2
  

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

• 33,5µl of pure water
  

• 0,5µl TaqPol
  

Program:

• Denat: 3min 94°C

• 5x:

Denat: 45sec 94°C

Anneal:45sec 53°C

Extend:45sec 72°C

• 25x:

Denat: 45sec 60°C

Anneal:45sec 60°C

Extend:45sec 72°C

• Final Extend: 10min 72°C

Template batches:

• TEV mutagenesis fragment I fraction I and TEV mutagenesis fragment II fraction I - fraction 1
  

• TEV mutagenesis fragment I fraction I and TEV mutagenesis fragment II fraction II - fraction 2
  

• TEV mutagenesis fragment I fraction I and TEV mutagenesis fragment II fraction III - fraction 3
  

• TEV mutagenesis fragment I fraction I and TEV mutagenesis fragment II fraction IV - fraction 4
  

• TEV mutagenesis fragment I fraction II and TEV mutagenesis fragment II fraction I - fraction 5
  

• TEV mutagenesis fragment I fraction II and TEV mutagenesis fragment II fraction II - fraction 6
  

• TEV mutagenesis fragment I fraction II and TEV mutagenesis fragment II fraction III - fraction 7
  

• TEV mutagenesis fragment I fraction II and TEV mutagenesis fragment II fraction IV - fraction 8
  

• TEV mutagenesis fragment I fraction III and TEV mutagenesis fragment II fraction I - fraction 13
  

• TEV mutagenesis fragment I fraction III and TEV mutagenesis fragment II fraction II - fraction 14
  

• TEV mutagenesis fragment I fraction III and TEV mutagenesis fragment II fraction III - fraction 15
  

• TEV mutagenesis fragment I fraction IV and TEV mutagenesis fragment II fraction IV - fraction 16
  

• TEV mutagenesis fragment I fraction IV and TEV mutagenesis fragment II fraction I - fraction 9
  

• TEV mutagenesis fragment I fraction IV and TEV mutagenesis fragment II fraction II - fraction 10
  

• TEV mutagenesis fragment I fraction IV and TEV mutagenesis fragment II fraction III - fraction 11
  

• TEV mutagenesis fragment I fraction IV and TEV mutagenesis fragment II fraction IV - fraction 12
  

Results:

• 16 different assembled TEV protease fractions with side directed mutagenesis to remove iGEM RS in nucleotide sequence
  

Further tasks:

• control of fragments with analytical agarose gelelctrophoresis
  

• PCR purification of fractions with the complete assembled TEV-Protease
  

• digest of TEV protease fractions with restriction enzymes NgoMIV and PstI and ligation with AraC into the digested (with EcoRI and PstI) vector pUP_SG1_ssTorA_CS-TEV_bla_AraC-TEV
  

• transformation of competent E.coli XL1 blue cells with ligated fragments, picking clones, colony PCR, mini-prep of plasmid, sequencing plasmid and survival test with positiv clones
  

Output:

• 5 assembled TEV proteases
  

• • TEV batch 2 c=
    

• • TEV batch 3 c=
    

• • TEV batch 7 c=
    

• • TEV batch 10 c=
    

• • TEV batch 16 c=
    

72th Labday 2011-08-22

Elimination of ampicillin resistance in pARW089

Investigators: Nadine, Jessica, Steffi, Nicole

Time: 2011-08-22

Aim:

• generation of pUP089 from pARW089, vector without ampicillin resistance

• for mdnA library and later screening

idea:

• digest w/ AvaII and religation

New primer ordered for cloning of geneIII into pARW089 (without mdnA)

Investigators: Sabine

Aim:

• generation of pARW089 containing only geneIII but not mdnA gene

• mdnA library could cloned into this vector for screening
  

Time: 16:00-16:35

Material/Method:

• Geneious

Result:

• new forward primer for geneIII containing NarI and NgoMIV restriction site and myc

• PCR product geneIII can cloned with NarI and AatII into pARWo89

Further tasks:

• PCR, digestion and ligation

Digestion of mdnA/geneIII fusion gene for cloning into pARW089 (2 step ligation)

Investigator: Sabine

Aim: cloning of mdnA/geneIII fusion gene into pARW089

Time: 2011-08-22, 16:30-17:30

Material/Method:

• 30 µl / 500 ng mdnA/geneIII

• 2 µl restriction enzyme NarI

• 1 µl restriction enzyme AatII

• 4 µl NEB 10x buffer 4

• 3 µl water

• 1 h, 37°C

Further Tasks:

• ligation into pARW089

Digestion of mdnA/geneIII fusion gene for cloning into pSB1C3 (2 step ligation)

Investigator: Sabine

Aim: cloning of mdnA/geneIII fusion gene into pSB1C3

Time: 2011-08-22, 16:30-17:30

Material/Method:

• 30 µl / 500 ng geneIII

• 1 µl restriction enzyme XbaI

• 1 µl restriction enzyme PstI

• 4 µl NEB 10x buffer 2

• 4 µl water

• 0,4 µl BSA

• 1 h, 37°C

Further Tasks:

• ligation into pSB1C3

Ligation of mdnA/geneIII into pARW089

Investigator: Sabine

Time: 2011-08-22, 18:00-19:00

Material/Method:

• 10 µl pARW089/NarI+AatII (60 ng/µl)

• 8 µl mdnA/geneIII (16 ng/µl)

• 2 µl 10x T4 ligase buffer

• 1 µl T4 ligase

• 1 h, room temperature

Further Tasks:

• transformation

Ligation of mdnA/geneIII into pSB1C3

Investigator: Sabine

Time: 2011-08-22, 18:00-19:00

Material/Method:

• 8 µl pARW089/NarI+AatII (60 ng/µl)

• 9 µl mdnA/geneIII (16 ng/µl)

• 2 µl 10x T4 ligase buffer

• 1 µl T4 ligase

• 1 h, room temperature

Further Tasks:

• transformation

Transformation of generated vector constructs in E. coli

Investigators: Sabine

Aim: amplification and control of generated vectors

• mdnA, geneIII or mdnA/geneIII in pSB1C3

• mdnA or mdnA/geneIII in pARW089

Time: 20:30-22:00

Method:

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

• incubation 15 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 kanamycin (pARW089) or chloramphenicol (pPSB1C3)
  

• storage over night at 37°C
  

Further tasks:
control cell clones

Digest of pSB1C3, pSB1T3 clones containing CFP and YFP, respectively as well as PCR products of promoters

Investigators: Jessica

Time: 2011-08-22

Materials

• pSB1C3 (BBa_K40304_Affibody_MiddleLinker) from

• pSB1T3+YFP I clone b and pSB1T3+YFP II clone a from 2011-08-21 (Katharina)

• PCR fragments: Arabinose, IPTG-inducible, constitutive

Method:

• Reaction mix:

	pSB1C3	pSB1T3+YFP I clone b	pSB1T3+YFP II clone a	PCR fragments
DNA	6	3	3	20
10x Buffer 4 NEB	2	2	2	3
XbaI	1	1	1	1
EcoRI	1	1	1	1
100x BSA	0.2	0.2	0.2	0.3
H2O	9.8	12.8	12.8	4.8
total	20	20	20	30

• at 37°C for approx. 3 h

• heat inactivation at 65°C for 20 min

Agarose gel

Gel extraction:

• using Promega- Wizard SV Gel and PCR Clean Up System

• elute in 30 µl nuclease-free water

Dephosphorylation of pSB1C3:

• using Promega TSAP

• adding 3 µl Buffer and 1 µl TSAP

• incubate at 37°C for 15 min

• heat inactivaton at 74°C for 15 min

Results:

73th Labday 2011-08-23

overnight culture of picked E. coli clones transformed with pPDV

Investigators: Sabine

Aim: amplification and purification of generated phage display vector pPDV089 for test digestion and sequencing

Time: 11:00-13.00

Method/Materials:

• 10 clones pARW089+mdnA/geneIII from 3 ragment ligation

• 10 clones pARW089+mdnA/geneIII from 2 step ligation

• 5 clones pARW089+mdnA

• 5 clones pSB1C3+mdnA

• 5 clones pSB1C3+geneIII

• 5 clones pSB1C3+mdnAIgeneIII

• 5 ml LB medium per clone containining 25 µg/ml chloramphenicol (pSB1C3) or kanamycin (pARW089)

• storage over night at 37°C and 800 rpm
  

Further tasks:

plasmid preparation, test digestion and sequencing

PCR: BioBrick mdnABC, mdnC, mdnE, mdnDE, mdnBC

Time: 2011-08-23, 8:00-11:30

Investigators: Nadine, Nicole

Materials

• vector: pARW089, 20.8.11, 333.4 ng/µl

• Phusion HF Polymerase, NEB

• Phusion HF Buffer

• dNTPs

• water

• Primer:

#	Primer
57	pf_mdnABC_EcoRI_NotI_XbaI 12.08.
58	pf_mdnB_EcoRI_NotI_XbaI 12.08.
59	pf_mdnC_EcoRI_NotI_XbaI 12.08.
60	pf_mdnD_EcoRI_NotI_XbaI 12.08.
61	pf_mdnE_EcoRI_NotI_XbaI 12.08.
62	pr_mdnABC_SpeI_NotI_PstI 12.08.
63	pr_mdnB_SpeI_NotI_PstI 12.08.
64	pr_mdnD_SpeI_NotI_PstI 12.08.
65	pr_mdnE_SpeI_NotI_PstI 12.08.

Protocol:

• Reaction mix

• • 2 µl pARW089 (diluted 1:100)

• • 1 µl dNTPs (10 mM)

• • 2.5 µl Primer forward (10 mM)

• • 2.5 µl Primer backward (10 mM)

• • 10 µl Buffer (5x)

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

• • 31.5 µl water

• • total volume: 50 µl

• Master mix

• • 12 µl pARW089 (diluted 1:100)

• • 6 µl dNTPs (10 mM)

• • 60 µl Buffer (5x)

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

• • 189 µl water

• Primer

• • mdnABC: 57, 62

• • mdnC: 59, 62

• • mdnE: 61, 65

• • mdnDE: 61, 64

• • mdnBC: 58, 62

2. PCR programs

• IGBIO02 for mdnABC, mdnC

• 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	59°C	30 s
Extension	72°C	20 s
DenaturationII	98°C	10 s
AnnealingII	72°C	30 s
ExtensionII	72°C	20 s
Final extension	72°C	10 min

• IGBIO03 for mdnE, mdnDE, mdnBC

• 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	52°C	30 s
Extension	72°C	60 s
DenaturationII	98°C	10 s
AnnealingII	72°C	30 s
ExtensionII	72°C	60 s
Final extension	72°C	10 min

Results:

• Output:

• • • mdnABC, Nad & Nic, 23.8.11

• • • mdnC, Nad & Nic, 23.8.11

• • • mdnE, Nad & Nic, 23.8.11

• • • mdnDE, Nad & Nic, 23.8.11

• • • mdnBC, Nad & Nic, 23.8.11

Digest of pSB1T3+YFPI/II vectors for ligation w/ promotors

Time: 2011-08-23, 9:00-

Investigators: Nadine, Nicole, Katharina

Materials

• vectors from 2011-08-21 (Katharina):

• • pSB1T3+YFP I clone b: 690.6 ng/µl

• • pSB1T3+YFP I clone c: 576.3 ng/µl

• • pSB1T3+YFP I clone a: 646.9 ng/µl

• • pSB1T3+YFP II plate 1 clone b: 599.8 ng/µl

• • pSB1T3+YFP II clone a: 562.8 ng/µl

• • pSB1T3+YFP II plate 2 clone d: 569.1 ng/µl

• • pSB1T3+YFP II plate 2 clone c: 415.0 ng/µl

• • pSB1T3+YFP I clone d: 559.9 ng/µl

• EcoRI HF

• XbaI

• BSA (10x)

• Buffer 4

• water

Protocol:

• Reaction mix

• • 2 µl DNA

• • 1 µl EcoRI

• • 1 µl XbaI

• • 0.3 µl BSA

• • 3 µl Buffer 4 (10x)

• • 22.7 µl water

• • total volume: 30 µl

• incubation at 37°C for 2 hrs

Agarose gel

Investigator: Jessica

• Gel extraction using NucleoSpin ExtractII (Macherey-Nagel)

• • no excision of pSB1T3+YFP II plate 2 clone c because of no visible band

Results:

Digest of amplified 14_3C protease (assembled fragments after sidedirected mutagenesis), amplified AraC (from pBAD_iGEM_express mVenus), plasmid pJC354_ssTorA_XhoI_CS-143C_NheI_blaFL and plasmid pJC354_ssTorA_XhoI_CS-TEV_NheI_blaFL

Investigator: Sebastian, Sascha

Material:

• Restriction enzymes NgoMIV, HindIII, BamHI, PstI-HF, EcoRI-HF (purchased from NEB)
  

• digest buffer 2 and 4 (puchased from NEB)
  

• BSA (purchased from NEB)
  

• plasmid pJC354_ssTorA_XhoI_CS-143C_NheI_blaFL
  

• plasmid pJC354_ssTorA_XhoI_CS-TEV_NheI_blaFL
  

• PCR amplified AraC-fragment
  

• PCR amplified and mutated 14_3C protease
  

Methode:

Standard protocoll for DNA digest.

Reaction batches:

• Batch 1: 21.7 µl 14_3C fraction I (c=157.0 ng/µl), 2.5 µl NgoMIV, 2.5 µl BamHI, 0.3 µl BSA, 3 µl Buffer 4
  

• Batch 2: 21.7 µl 14_3C fraction II(c=157.5 ng/µl), 2.5 µl NgoMIV, 2.5 µl BamHI, 0.3 µl BSA, 3 µl Buffer 4
  

• Batch 3: 22.0 µl AraC (c=175.0 ng/µl), 2.5 µl NgoMIV, 2.5 µl HindIII, 3 µl Buffer 2
  

• Batch 4: 22.0 µl AraC (c=175.0 ng/µl), 2.5 µl NgoMIV, 2.5 µl EcoRI-HF, 3 µl Buffer 4
  

• Batch 5: 6,9 µl plasmid pJC354_ssTorA_XhoI_CS-143C_NheI_blaFL II (c= ng/µl), 1.0 µl HindIII, 2.5 µl BamHI, 0.1 µl BSA, 1 µl Buffer 2
  

• Batch 6: 6,9 µl plasmid pJC354_ssTorA_XhoI_CS-143C_NheI_blaFL VII (c= ng/µl), 1.0 µl HindIII, 2.5 µl BamHI, 0.1 µl BSA, 1 µl Buffer 2

• Batch 7: 7.0 µl plasmid pJC354_ssTorA_XhoI_CS-TEV_NheI_blaFL (c= ng/µl), 1.0 µl EcoRI-HF, 1.0 µl PstI-HF, 1.0 µl Buffer 4
  

Further tasks:

All plasmid fractions should be purified with 1.0% agarose gel, all other fragments with 1.5% agarose gel and extracted with Macherey-Nagel Nucleo SpinII Kit.

• Ligation of fragments in different combinations and transformation of competent E.coli XL1 blue cells.
  

Output:

• digested fragments of AraC, 14_3C protease and plasmid backbones for transformation (AraC + 14_3C + "14_3C-backbone", AraC + TEV + "TEV-backbone")
  

1) EcoRI_AraC_NgoMIV

2) HindIII_AraC_NgoMIV

3) NgoMIV_14_3C_BamHI Batch 1

4) NgoMIV_14_3C_BamHI Batch 2

5) BamHI_pJC354_ssTorA_XhoI_CS-143C_NheI_blaFL_HindIII Batch 1

6) BamHI_pJC354_ssTorA_XhoI_CS-143C_NheI_blaFL_HindIII Batch 2

7) BamHI_pJC354_ssTorA_XhoI_CS-TEV_NheI_blaFL_HindIII

Agarose Gel of PCR products for BioBricks mdnABC, mdnBC, mdnC, mdnC, mdnD, mdnDE, mdnE and PCR purification

Investigators: Nadine, Nicole, Katharina, Jessica

Time: 2011-08-23, 13:00-16:00

Materials:

• PCR products

• Agarose broad range (Roth)

• 1x TAE buffer

• Gel Red

• DNA Ladder Mix (Fermentas)

• 6x Loading Dye (Fermentas)

Production of one 1 % agarose gel

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

• Adding 2 µl gel red to each gel

Loading gels and running

• Add µl Loading dye to each 2 µl sample

• 12 µl DNA Ladder Mix

• Running conditions: 110 V, approx. 45 min

Loading of gel

lane	Sample	Volume in µl	Expected size in bp
M	marker	12
1	mdnABC	-	2492
2	mdnB	-	1021
3	mdnBC	-	2045
4	mdnC	-	1007
5	mdnD	-	600
6	mdnDE	-	2800
7	mdnE	-	2100

Result gel 1:

• all PCR products appear as expected, except mdnB

• purification of PCR products w/ Machery and Nagel PCR purification kit

• Output:

• • PCR pur., mdnABC, 48.1 ng/µl, Kat, 23.8.11

• • PCR pur., mdnBC, 70.1 ng/µl, Kat, 23.8.11

• • PCR pur., mdnC, 62 ng/µl, Kat, 23.8.11

• • PCR pur., mdnD, 67.1 ng/µl, Kat, 23.8.11

• • PCR pur., mdnDE, 61.8 ng/µl, Kat, 23.8.11

• • PCR pur., mdnE, 68.7 ng/µl, Kat, 23.8.11

Further task:

• repeat PCR for mdnB

• digestion of other PCR products

• ligation w/ pSB1C3

Test digest of pSB1A3+CFP/YFP and pSB1K3+CFP/YFP vectors w/ promotors

Investigators: Steffi, Nadine, Nicole

Materials

• vectors from 2011-08-20 (Katharina):

• • pSB1A3-CFP +Ara (clone A) 12: 342.1 ng/µl

• • pSB1A3-CFP +Ara (clone B) 7: 252.5 ng/µl

• • pSB1A3-CFP +Ara (clone C) 6: 356.9 ng/µl

• • pSB1A3-YFP +Ara (clone A) 1: 339.2 ng/µl

• • pSB1A3-YFP +Ara (clone B) 5: 379.0 ng/µl

• • pSB1A3-YFP +Ara (clone C) 14: 221.4 ng/µl

• • pSB1A3-CFP +Lac (clone A) 15: 319.3 ng/µl

• • pSB1A3-CFP +Lac (clone B) 34: 105.5 ng/µl

• • pSB1A3-CFP +Lac (clone C) 33: 135.7 ng/µl

• • pSB1A3-YFP +Lac (clone A) 16: 110.6 ng/µl

• • pSB1A3-YFP +Lac (clone B) 4: 340.4 ng/µl

• • pSB1A3-YFP +Lac (clone C) 13: 290.6 ng/µl

• • pSB1A3-CFP + const (clone A) 11: 312.2 ng/µl

• • pSB1A3-CFP + const (clone B) 9: 218.0 ng/µl

• • pSB1A3-CFP + const (clone C) 10: 201.7 ng/µl

• • pSB1A3-YFP + const (clone C) 28: 238.0 ng/µl

• • pSB1K3-CFP +Ara (clone A) 25: 175.2 ng/µl

• • pSB1K3-CFP +Ara (clone B) 31: 190.2 ng/µl

• • pSB1K3-CFP +Ara (clone C) 24: 193.8 ng/µl

• • pSB1K3-YFP +Ara (clone A) 22: 119.6 ng/µl

• • pSB1K3-YFP +Ara (clone B) 19: 149.4 ng/µl

• • pSB1K3-YFP +Ara (clone C) 30: 135.1 ng/µl

• • pSB1K3-CFP +Lac (clone A) 20: 138.4 ng/µl

• • pSB1K3-CFP +Lac (clone B) 21: 58.3 ng/µl

• • pSB1K3-CFP +Lac (clone C) 23: 122.0 ng/µl

• • pSB1K3-YFP +Lac (clone A) 29: 69.3 ng/µl

• • pSB1K3-YFP +Lac (clone B) 17: 153.1 ng/µl

• • pSB1K3-YFP +Lac (clone C) 18: 90.9 ng/µl

• • pSB1K3-CFP + const (clone A) 26: 89.8 ng/µl

• • pSB1K3-CFP +const (clone B) 8: 252.5 ng/µl

• • pSB1K3-CFP +const (clone C) 2: 256.6 ng/µl

• • pSB1K3-YFP + const (clone A) 27: 55.7 ng/µl

• • pSB1K3-YFP +const (clone B) 3: 231.9 ng/µl

• • pSB1K3-YFP + const (clone C) 32: 174.9 ng/µl
    

• BamHI

• AatII

• HpaI

• HincII

• NotI

• BSA (10x)

• Buffer 3

• Buffer 4

• water

Protocol:

• Reaction mix (Arabinose)

• • 0.5 µl DNA

• • 1 µl BamHI

• • 1 µl AatII

• • 0.3 µl BSA

• • 3 µl Buffer 4 (10x)

• • 24.2 µl water

• Reaction mix (Lac)

• • 0.5 µl DNA

• • 1 µl HpaI

• • 1 µl AatII

• • 3 µl Buffer 4 (10x)

• • 24.5 µl water

• Reaction mix (constitutive)

• • 0.5 µl DNA

• • 1 µl HincII

• • 1 µl NotI

• • 0.3 µl BSA

• • 3 µl Buffer 3 (10x)

• • 24.2 µl water

• • total volume each: 30 µl

• incubation at 37°C for 1 hrs

further tasks:

• gel electrophoresis

• ligation

• transformation

Agarose Gel of pSB1A3+CFP/YFP and pSB1K3+CFP/YFP

Investigators: Steffi, Katharina, Nadine

Materials:

• digestion products

• Agarose broad range (Roth)

• 1x TAE buffer

• Gel Red

• DNA Ladder Mix (Fermentas)

• 6x Loading Dye (Fermentas)

Production of one 1 % agarose gel

• 1 x 1 % gel: 1,0 g agarose in 100 ml 1x TAE buffer

• Adding 4 µl gel red to gel

Loading gels and running

• Add 5 µl Loading dye to each 2 µl sample

• 7 µl DNA Ladder Mix

• Running conditions: 110 V, approx. 45 min

Loading of gel

lane	Sample	Volume in µl	Expected size in bp
M	marker	7
1	pSB1A3-CFP +Ara (clone A)	15
2	pSB1A3-CFP +Ara (clone B)	15
3	pSB1A3-CFP +Ara (clone C)	15
4	pSB1A3-YFP +Ara (clone A)	15
5	pSB1A3-YFP +Ara (clone B)	15
6	pSB1A3-YFP +Ara (clone C)	15
7	pSB1K3-CFP +Ara (clone A)	15
8	pSB1K3-CFP +Ara (clone B)	15
9	pSB1K3-CFP +Ara (clone C)	15
10	pSB1K3-YFP +Ara (clone A)	15
11	pSB1K3-YFP +Ara (clone B)	15
12	pSB1K3-YFP +Ara (clone C)	15
M	marker	7
13	pSB1A3-CFP + const (clone A)	15
14	pSB1A3-CFP + const (clone B)	15
15	pSB1A3-CFP + const (clone C)	15
16	pSB1A3-YFP + const (clone C)	15
17	pSB1K3-CFP + const (clone A)	15
18	pSB1K3-CFP + const (clone B)	15
19	pSB1K3-CFP + const (clone C)	15
20	pSB1K3-YFP + const (clone A)	15
21	pSB1K3-YFP + const (clone B)	15
22	pSB1K3-YFP + const (clone C)	15
M	marker	7
23	pSB1A3-CFP +Lac (clone A)	15
24	pSB1A3-CFP +Lac (clone B)	15
25	pSB1A3-CFP +Lac (clone C)	15
26	pSB1A3-YFP +Lac (clone A)	15
27	pSB1A3-YFP +Lac (clone B)	15
28	pSB1A3-YFP +Lac (clone C)	15
29	pSB1K3-CFP +Lac (clone A)	15
30	pSB1K3-CFP +Lac (clone B)	15
31	pSB1K3-CFP +Lac (clone C)	15
32	pSB1K3-YFP +Lac (clone A)	15
33	pSB1K3-YFP +Lac (clone B)	15
34	pSB1K3-YFP +Lac (clone C)	15

Result gel:

• sample ... used for sequencing

further tasks:

• sequencing

Agarose Gel of pARW089 Ava II A (3µl DNA)/ B (4µl DNA) (= pUP089)

Investigators: Niels, Nadja

Materials:

• digestion products

• Agarose broad range (Roth)

• 1x TAE buffer

• Gel Red

• DNA Ladder Mix (Fermentas)

• 6x Loading Dye (Fermentas)

Production of one 0,8 % agarose gel

• 1 x 0,8 % gel: 0,4 g agarose in 50ml 1x TAE buffer

• Adding 2 µl gel red to gel

Loading gels and running

• Add 6 µl Loading dye to each 30 µl sample

• 20 µl DNA Ladder Mix 1:10 diluted

• Running conditions: 100 V, approx. 50 min

Loading of gel

lane	Sample	Volume in µl	Expected size in bp
M	marker	20
1	pARW089 Ava II A	36	over 10kbp
2	pARW089 Ava II B (4µl DNA)	36	over 10kbp

Result gel:

• sample used for gel extraction (Promega Kit Wizard SV Gel and PCR Clean Up System)

• sample used for ligation

Further tasks:

• transformation

Ligation of pSB1C3 and pSB1T3-YFPI/II with promoters and ligation of pARW089 AvaII

Investigators: Jessica

Materials:

• T4 Ligase (Promega)

• 10x T4 Ligase Buffer (Promega)

• digested, gel purified, dephoshorylated vectors: pSB1C3, pSB1T3-YFP I clone c, pSB1T3-YFP II plate 2 clone d

• Inserts: Ara promoter, Lac promoter, constitutive promoter

Method:

• 10 µl reaction for pSB vectors:

• • 1 µl 10x T4 Ligase Buffer

• • 1 µl T4 Ligase

• • 6 µl vector

• • 2 µl insert

• 10 µl reaction for pARW089 vector:

• • 1 µl 10x T4 Ligase Buffer

• • 1 µl T4 Ligase

• • 8 µl vector

• at 14°C overnight

Results:

• 14 ligation products

• • pSB1C3 + Ara

• • pSB1C3 + Lac

• • pSB1C3 + const

• • pSB1C3 + H₂O

• • pSB1T3-YFP II + Ara

• • pSB1T3-YFP II + Lac

• • pSB1T3-YFP II + const

• • pSB1T3-YFP II + H₂O

• • pSB1T3-YFP I + Ara

• • pSB1T3-YFP I + Lac

• • pSB1T3-YFP I + const

• • pSB1T3-YFP I + H₂O
    

• • pARW089 AvaII A = pUP089 A

• • pARW089 AvaII B = pUP089 B

Further tasks:

• transformation

Over night culture of several pSB1A3+CFP/YFP and pSB1K3+CFP/YFP with promotor

Time: 2011-08-23, 18:00-22:30

Investigators: Niels, Jessica, Nadja

Materials

• 5 ml LB media

• 5 µl antibotic (KAN/AMP - Stock)

• clone

• • KAN YFP ava clone IV (from plate)

• • KAN YFP ava clone V (from plate)

• • KAN YFP ava clone VI (from plate)

• • KAN CFP const clone I (from pre-culture)

• • KAN CFP const clone II (from pre-culture)

• • KAN CFP const clone III (from pre-culture)

• • KAN YFP const clone I (from pre-culture)

• • KAN YFP const clone II (from pre-culture)

• • KAN YFP const clone III (from pre-culture)

• • AMP CFP const clone I (from pre-culture)

• • AMP CFP const clone II (from pre-culture)

• • AMP CFP const clone III (from pre-culture)

• • AMP YFP const clone I (from pre-culture)

conditions

• 37 °C

• 250 rpm

• over night

further tasks:

• miniprep

• digest

• analytic gel

sequencing of

Time: 2011-08-23, 18:00-22:30

Investigators: Nadja ,Niels ,Jessica

Materials

Sequencing by eurofins mwg|operon

Label - Barcode

• 1cB - AKM001W053

• 1cC - AKM001W054

• 1cA - AKM001W055

• 2cB - AKM001W056

• 2cA - AKM001W057

• 2cD - AKM001W058

• 2cC - AKM001W059

• 1cD - AKM001W060

sequencing of

Time: 2011-08-23, 18:00-22:30

Investigators: Nadja ,Niels ,Jessica

Materials

Sequencing by eurofins mwg|operon

Label - Barcode

• K3CAcA - AKM001W061

• A3CAcA - AKM001W062

• A3YAcA - AKM001W063

• K3YIcC - AKM001W064

• K3CIcB - AKM001W065

• A3YIcB - AKM001W066

• A3cIcA - AKM001W067

over night culture

Time: 2011-08-23, 18:00-22:30

Investigators: Niels, Jessica, Nadja

Materials

• 5 ml LB Media

• 5 µl Amp/Kan

• clones

• • pSBA3-YFP+const

• • pSBA3-CFP+const A

• • pSBA3-CFP+const C

• • pSBK3-YFP+const A

• • pSBK3-CFP+const A

• • pSBK3-YFP+ara V

overnight 37°C

further tasks:

• miniprep

74th Labday 2011-08-24

Transformation w/ pUP089 A and B, BBa_K40304_Affibody_MiddleLinker+Ara/Const/Lac, pSB1T3+YFPI+Ara/Const/Lac and pSB1T3+YFPII+Ara/Const/Lac

Investigators: Nicole, Nadine

Time: 7:00-9:00

Aim:

• creating pARW089 without Amp resistance, called then pUP089

• get expression backbones w/ Tet or Cm resistance and Ara, Const. or Lac promotors

Material:

• XL1-blue (competent)

• RV-308 (competent)

• ligation products from o.n. ligation from 2011-08-23 (Jessica)

• • pSB1C3 + Ara

• • pSB1C3 + Lac

• • pSB1C3 + const

• • pSB1C3 + H₂O

• • pSB1T3-YFP II + Ara

• • pSB1T3-YFP II + Lac

• • pSB1T3-YFP II + const

• • pSB1T3-YFP II + H₂O

• • pSB1T3-YFP I + Ara

• • pSB1T3-YFP I + Lac

• • pSB1T3-YFP I + const

• • pSB1T3-YFP I + H₂O
    

• • pARW089 AvaII A = pUP089 A

• • pARW089 AvaII B = pUP089 B

• for pSB1T3 plasmids RV-308 cells were used

• for others: Xl1-blue

• LB-Agar

• tetracycline

• chloramphenicol

• kanamycine

Method:

• addition of 2 µl ligation reaction to XL1-blue cells
  

• incubation 60 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:

• • pUP089

• • • 100 µg/ml ampicilin
      

• • • 100 µg/ml kanamycin
      

• • • 100 µg/ml ampicilin and kanamycin
      

• • pSB1T3

• • • 100 µg/ml tetracyclin

• • pSB1C3

• • • 100 µg/ml chloramphenicol

• incubation for 20 hrs at 37°C
  

Further tasks:
control cell clones

Miniprep of E.coli overnight culture containing created pPDV089

Investigators: Sabine, Laura

Aim: purification of pPDV089 for test digestion and sequencing

Time: 10:00-15:00

Method/Materials:

• see protocol 5.1 of the NucleoSpin Plasmid Kit
  

• 5 clones containing pSB1C3+mdnA

• 5 clones containing pSB1C3-geneIII

• 5 clones containing pSB1C3-mdnA/geneIII

• 10 clones containing pARW089+mdnA

• 10 clones containing pARW089+mdnA/geneIII (3 fragment ligation)

• 10 clones containing pARW089+mdnA/geneIII (2 step ligation)

Further tasks: test digestion

Test digestion of purified plasmids pARW089

Investigators: Sabine

Aim:control of liagation of mdnA and geneIII into pARW089

Time: 13:00-17:00

Materials/Methods:

• 0,5 µl XbaI

• 0,5 µl SpeI

• 2 µl 10x buffer 4 (NEB)

• 0,2 µl BSA

• 2 µl vector DNA (ca. 1 µg)

• 14,8 µl water

• incubate for 3 h at 37°C

Results:

• expected band sizes for pARW089 containing mdnA and geneIII: ca. 5300 bp, 4500 bp, 630 bp and 90

• expected band sizes for pARW089 not containing the inserts: ca. 10000 bp, 630 bp and 90 bp

Test digestion of purified plasmids pSB1C3

Investigators: Sabine

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

Time: 18:00-18:30

Materials/Methods:

• per reaction:

• 0,5 µl XbaI

• 0,5 µl PvuI

• 2 µl 10x buffer 3 (NEB)

• 0,2 µl BSA

• 5 µl vector DNA (ca. 1 µg)

• 11,8 µl water

• incubate over night at 37°C

Results:

Agarose gel electrophoresis of digested mdn fragments and pSB1C3, gel extraction and ligation

Investigators: Nicole, Jessica, Steffi, Niels

Time: 2011-08-24,

Materials

• digests of :

• • mdnB

• • mdnC

• • mdnD

• • mdnE

• • mdnBC

• • mdnDE

• • mdnABC

• • pSB1C3

• agarose, 1x TAE, gel red

• gel extraction kit (Promega)

• thermosensitive alkaline phoshatase (TSAP, Promega)

• ligase

Gel electrophoresis:

• 1,25% agarose gel (0.625 g agarose + 50 ml 1x TAE, 2 µl gel red)

• 30 µl digest + 6 µl 6x loading dye
  

Gel 1

lane	Sample	Volume in µl	Expected size in bp
1	mdnB	15	~1021
2	mdnB	15	~1021
3	mdnC	15	~1007
4	mdnC	15	~1007
5	mdnD	15	~600
6	mdnD	15	~600
M	GeneRuler™ DNA Ladder Mix (diluted 1:10)	10

Gel 2

lane	Sample	Volume in µl	Expected size in bp
1	mdnE	36	~2100
2	mdnBC	36	~2045
3	mdnDE	36	~2800
4	mdnABC	36	~2492
5	-
6	pSB1C3	36	~2389, 243

Gel extraction:

• using Promega- Wizard SV Gel and PCR Clean Up System

Dephosphorylation of pSB1C3:

• using Promega TSAP

• • adding 5 µl Buffer and 1 µl TSAP

• • incubate at 37°C for 15 min

• • heat inactivaton at 74°C for 15 min
    

Nanodrop

sample	c [ng/µl]	260nm/280nm	260nm/230nm
mdnB	10,2	2,08	0,57
mdnC	13,4	2,3	0,4
mdnD	11,8	1,94	0,4
mdnE	13,3	1,93	0,78
mdnBC	16,3	1,91	0,79
mdnDE	13,8	1,92	0,60
mdnABC	13,4	1,89	0,68
C3	44,4	1,90	1,45

Ligation:

Insert	[µl]	Vector	[µl]
mdnB	6	C3	2
mdnC	6	C3	2
mdnD	7	C3	1
mdnE	5	C3	3
mdnBC	5	C3	3
mdnDE	5	C3	3
mdnABC	5	C3	3

• 1 µl 10x Buffer

• 1 µl T4 Ligase

• • over night by 14°C

further tasks:

• transformation

Digest of pSB1A3+CFP/YFP and pSB1K3+CFP/YFP vectors w/ promotors

Investigators: Jessica, Nadja

Time: 2011-08-24, 14:00-20:00

Materials

• vectors from 2011-08-24:

• • pSB1A3-CFP + const (clone A)

• • pSB1A3-CFP + const (clone B)

• • pSB1A3-CFP + const (clone C)

• • pSB1A3-YFP + const (clone C)

• • pSB1K3-YFP +Ara (clone V)

• • pSB1K3-YFP +Ara (clone V box)

• • pSB1K3-YFP +Ara (clone VI)

• • pSB1K3-CFP + const (clone A)

• • pSB1K3-CFP +const (clone B)

• • pSB1K3-CFP +const (clone C)

• • pSB1K3-YFP + const (clone A)

• • pSB1K3-YFP +const (clone B)

• • pSB1K3-YFP + const (clone C)
    

• NdeI

• NruI

• HincII

• NotI

• BSA (100x)

• Buffer 3

• water
  

Digest:

• Reaction mix (Arabinose)

• • 0.5 µl DNA

• • 0.5 µl NdeI

• • 0.5 µl NruI

• • 2 µl Buffer 3 (10x)

• • 16.5 µl water

• Reaction mix (constitutive)

• • 0.5 µl DNA

• • 0.5 µl HincII

• • 0.5 µl NotI

• • 0.2 µl BSA

• • 2 µl Buffer 3 (10x)

• • 16.3 µl water

• total volume each: 20 µl

• incubation at 37°C for 1.5 hrs
  

Gel electrophoresis:

• 1% agarose gel (0.5 g agarose + 50 ml 1x TAE, 2 µl gel red)

• 20 µl digest + 4 µl 6x loading dye
  

Gel 1

lane	Sample	Volume in µl	Expected size in bp
M	GeneRuler™ DNA Ladder Mix (diluted 1:10)	20
1	pSB1A3-CFP + const (clone A) (undigested)	6
2	pSB1A3-CFP + const (clone A)	15	2145, 804
3	pSB1A3-CFP + const (clone B)	15	2145, 804
4	pSB1A3-CFP + const (clone C)	15	2145, 804
5	pSB1A3-YFP + const (clone C)	15	2145, 804
6	-
7	pSB1K3-YFP +Ara (clone V) undigested	6
8	pSB1K3-YFP +Ara (clone V)	15	~ 2800, 870, 430
9	pSB1K3-YFP +Ara (clone V box)	15	~ 2800, 870, 430
10	pSB1K3-YFP +Ara (clone VI)	15	~ 2800, 870, 430

Gel 2

lane	Sample	Volume in µl	Expected size in bp
M	GeneRuler™ DNA Ladder Mix (diluted 1:10)	20
1	-
2	pSB1K3-CFP + const (clone A)	15	2145, 804
3	pSB1K3-CFP +const (clone B)	15	2145, 804
4	pSB1K3-CFP +const (clone C)	15	2145, 804
5	pSB1K3-YFP + const (clone A)	15	2145, 804
6	pSB1K3-YFP +const (clone B)	15	2145, 804
7	pSB1K3-YFP + const (clone C)	15	2145, 804
8	pSB1K3-CFP + const (clone A) undigested	6

further tasks:

• repeat digest of pSB1K3 + const with more DNA

• have a look at digest of pSB1K3 + Ara

Transformation w/ pUP089 A and B, BBa_K40304_Affibody_MiddleLinker+Ara/Const/Lac, pSB1T3+YFPI+Ara/Const/Lac and pSB1T3+YFPII+Ara/Const/Lac

Investigators: Nicole, Nadine

Time: 7:00-9:00

Aim:

• creating pARW089 without Amp resistance, called then pUP089

• get expression backbones w/ Tet or Cm resistance and Ara, Const. or Lac promotors

Material:

• XL1-blue (competent)

• RV-308 (competent)

• ligation products from o.n. ligation from 2011-08-23 (Jessica)

• • pSB1C3 + Ara

• • pSB1C3 + Lac

• • pSB1C3 + const

• • pSB1C3 + H₂O

• • pSB1T3-YFP II + Ara

• • pSB1T3-YFP II + Lac

• • pSB1T3-YFP II + const

• • pSB1T3-YFP II + H₂O

• • pSB1T3-YFP I + Ara

• • pSB1T3-YFP I + Lac

• • pSB1T3-YFP I + const

• • pSB1T3-YFP I + H₂O
    

• • pARW089 AvaII A = pUP089 A

• • pARW089 AvaII B = pUP089 B

• for pSB1T3 plasmids RV-308 cells were used

• for others: Xl1-blue

• LB-Agar

• tetracycline

• chloramphenicol

• kanamycine

Method:

• addition of 2 µl ligation reaction to XL1-blue cells
  

• incubation 60 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:

• • pUP089

• • • 100 µg/ml ampicilin
      

• • • 100 µg/ml kanamycin
      

• • • 100 µg/ml ampicilin and kanamycin
      

• • pSB1T3

• • • 100 µg/ml tetracyclin

• • pSB1C3

• • • 100 µg/ml chloramphenicol

• incubation for 20 hrs at 37°C
  

Further tasks:
control cell clones

Miniprep of o. n. culture of pSB1K3 constitutive and Ara as well as pSB1A3 constitutive and Ara

Time: 7:00-

Investigators: Nicole, Nadine, Nadja

Results:

Concentration:

• pSBA3-YFP+const C = 470,8 ng/µl

• pSBA3-CFP+const A = 889,2 ng/µl

• pSBA3-CFP+const C = 692,8 ng/µl

• pSBK3-YFP+const A = 495,0 ng/µl

• pSBK3-CFP+const A = 1142,0 ng/µl

• pSBK3-YFP+ara V = 380,1 ng/µl
  

Further task:

• sequencing

75th Labday 2011-08-25

check plates from transformation (expression backbones in XL1-blue, 2011-08-24)

Time: 7:00-7:20

Investigators: Nicole, Nadine

Aim:

• build expression backbones w/ different promotors

Material:

• on chloramphenicol plates (in XL1 blue)

• • pSB1C3 + Ara

• • pSB1C3 + Lac

• • pSB1C3 + const

• • pSB1C3 + H₂O

• • pSB1T3-YFP II + Ara

• • pSB1T3-YFP II + Lac

• • pSB1T3-YFP II + const

• • pSB1T3-YFP II + H₂O

• • pSB1T3-YFP I + Ara

• • pSB1T3-YFP I + Lac

• • pSB1T3-YFP I + const

• • pSB1T3-YFP I + H₂O
    

Results:

• buffer control was over grown

conclusions:

• something is wrong with competent cells

Further tasks:

• check competent cells

check plates from transformation (pUP089 in XL1-blue, 2011-08-24)

Time: 7:00-7:20

Investigators: Nicole, Nadine

Aim:

• creating pARW089 without Amp resistance, called then pUP089

Material:

• pUP089 A in XL1 blue

• pUP089 B in XL1 blue

• on kan, amp or kan and amp plates

Results:

conclusions:

• elimination of ampicillin resistance has worked

• pUP089 vector will be used for mdnA library

Sequencing of pSB1K3 constitutive and Ara as well as pSB1A3 constitutive and Ara from 2011-08-24

Time: 19:00-

Investigators: Jessica, Nadja

Materials:

• pSBA3-YFP+const C = 470,8 ng/µl

• pSBA3-CFP+const A = 889,2 ng/µl

• pSBA3-CFP+const C = 692,8 ng/µl

• pSBK3-YFP+const A = 495,0 ng/µl

• pSBK3-CFP+const A = 1142,0 ng/µl

• pSBK3-YFP+ara V = 380,1 ng/µl

• Primer : VF2

• eurofins mwg/ operon Label-Barcodes
  

Method:

• 70µg/µl DNA, total volume of 15µl

• 2 pmol/µl Primer, ca. 10µl for each sample
  

Further task:

• analysing results

Transformation of pSB1C3 with several mdn genes/ promotor and pSB1T3-YFP I/II with several promotor

Investigators: Niels, Jessica

Material:

• XL1-blue (competent)

• RV-308 (competent)

• ligation products :

• • pSB1C3 + mdnB

• • pSB1C3 + mdnC

• • pSB1C3 + mdnD

• • pSB1C3 + mdnE

• • pSB1C3 + mdnBC

• • pSB1C3 + mdnABC

• • pSB1C3 + mdnDE

• • pSB1C3 + Ara

• • pSB1C3 + Lac

• • pSB1C3 + const

• • pSB1C3 + H₂O

• • pSB1T3-YFP II + Ara

• • pSB1T3-YFP II + Lac

• • pSB1T3-YFP II + const

• • pSB1T3-YFP II + H₂O

• • pSB1T3-YFP I + Ara

• • pSB1T3-YFP I + Lac

• • pSB1T3-YFP I + const

• • pSB1T3-YFP I + H₂O
    

• for pSB1T3 plasmids RV-308 cells were used

• for others: Xl1-blue

• LB-Agar

• tetracycline

• chloramphenicol

Method:

• addition of 2 µl ligation reaction to XL1-blue cells
  

• incubation 60 min on ice,
  

• heat shock 90 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:

• • pSB1T3

• • • 100 µg/ml tetracyclin

• • pSB1C3

• • • 100 µg/ml chloramphenicol

• incubation over night at 37°C
  

Further tasks:
control cell clones

Results (from 2011-08-26):

• for transformations of pSB1C3 + mdn fragments 2 clones were picked from each plate

• plates of pSB1C3 and pSB1T3 with promoters were thrown away because of too many colonies on control

PCR of complete mdn cluster for biobrick-construction

Time: 2011-08-25

Investigators: Nadine, Jessica

Material:

• pARW089, 20.8.11, 3.3 ng/µl

• pARW071, 27.5.11, 6.3 ng/µl

• dNTPs

• primer 77, 78, 79, 80

• HF Phusion Buffer 5x* Phusion Polymerase

Method:

• 2µl pARW089, 1µl pARW071, respectively

• 1µl dNTPs

• 2µl forward primer (10mM)

• 2µl reverse primer (10mM)

• 10µl HF Phusion Buffer 5x

• 0.5 µl Phusion Polymerase

• 31.5 µl water (total volume= 50µl)

• reaction

Step	Temperature	Time
Hot Start	98°C	Hold
Initial denaturation	98°C	30 sec
Denaturation	95°C	30 s
Annealing	59°C	60 s
Extension	72°C	2 min
DenaturationII	95°C	30 s
AnnealingII	59°C	60 s
ExtensionII	72°C	2 min
Final extension	68°C	5 min

Result:

• three different PCR samples

• • PCR sample 1: mdnABCDE from pARW071, Primer: 77/78

• • PCR sample 2: mdnABCDE with T7 from pARW089, Primer: 77/79

• • PCR sample 3: mdnABCDE without T7 from pARW089, Primer: 77/80

Output:

• mdnABCDE71

• mdnABCDE89-T7

• mdnABCDE89

Further Tasks:

• agarose gel

• PCR clean up

• restriction enzyme digestion

• ligation

• transformation

gel purification of AraC, TEV, 14_3C and TEv vector and 14_3C vector

Investigator: Stefan

Material:

• Gel purification kit (Machery & Nagel)

Methode:

protocol for gel purification.

Further tasks:

ligation of different fragmentsand transformation of competent E.coli XL1 blue cells.

ligation of AraC, TEV/14_3C with the TEV/14_3C vector

Investigator: Stefan

Material:

optional table caption

Column heading 1	TEV 1 µL	TEV 2 µL	TEV 3 µL	TEV 4 µL	14_3C 1 µL	14_3C 2 µL	control 14_3C vector µL	control TEV vector µL
AraC	0.8 (35 ng)	1.8 (13.5 ng)	1.8 (8.8 ng)	2.3 (8.8 ng)	3.4 (7.5 ng)	1.4 (35 ng)	0	0
Protease	1.4 (11.9 ng)	1.3 (6.3 ng)	0.3 (61.9 ng)	0.7 (20.2 ng)	1.8 (7.1 ng)	3.6 (61.9 ng)	0	0
vector	5.8 (6.4 ng)	4.9 (6.4 ng)	6 (5.2 ng)	5.1 (5.2 ng)	2.8 (11.6 ng)	3. (22.2 ng)	5.8 (6.4 ng)	4.9 (6.4 ng)
buffer	1	1	1	1	1	1	1	1
ligase	1	1	1	1	1	1	1	1
H2O	0	0	0	0	0	0	2.8	5.2

Method:

ligation at room temperatur for 1 h

Further tasks:

transformation in competent cells

transformation of ligations

Investigator: Stefan

Material:

Method:

streak out of LB plattes with CM, use 150 µL and spin down the rest and streak it out, too.

Further tasks:

checking clones

over night culture of pUP089 A/*B from 24.08.2011

Investigators: Niels, Nadja

Materials/Method:

• 5 ml LB media

• 5 µl Kanamycin (50mg/ml)

• • clone of

• • • pUP089 A - I

• • • pUP089 A - II

• • • pUP089 A - III

• • • pUP089 B - I

• • • pUP089 B - II

• • • pUP089 B - III

• 37°C, 250 rpm, over night

Further task:

• miniprep

PCR of geneIII and mdnA

Investigators: Sabine

Aim:

• amplification of mdnA and geneIII for cloning into pSB1C3
  

Reaction Components:

• 2 µl/ 20 ng geneIII (cut out from pARW089)

• 0,25 µl Taq Polymerase S (BioScience)

• 1 µl dNTPs

• 1 µl primer pf_geneIII_EheI_XbaI_NgoMIV

• 1 µl primer pr_geneIII_iGEM_AatII

• 5 µl 10x PCR Buffer S

• 39,75 µl water

• 5 µl/ 50 ng pak bla KDIR (mdnA)

• 0,25 µl Taq Polymerase S (BioScience)

• 1 µl dNTPs

• 1 µl primer pf_mdnA_iGEM_EheI

• 1 µl primer pr_mdnA_iGEM_AatII

• 5 µl 10x PCR Buffer S

• 36,75 µl water

Further tasks:

• analytic gel electrophoresis

• purification

• digestion

Analytic gel electrophoresis to control PCR

Investigators: Sabine

Time: 12:00-13:00

Aim: control of PCR

Material/Method:

• PCR products (mdnA and geneIII)

• 1 % agarose gel

• 1x TAE buffer

• Gel Red

• DNA Ladder Mix (1:10) (Fermentas)

• 6x Loading Dye (Fermentas)

• Gel Red

Result:

• PCR product with right band sizes

• mdnA ca. 200 bp

• geneIII ca. 500 bp

Further tasks:

• purification

• digestion

prepare samples for sequencing (created pPDV089)

Investigators: Sabine

Time: 12:00-13:00

Aim: sequencing (GATC)

Material/Method:

• purified plasmids of clones 3F6, 3F8, 2S7 and 2S14 (80 ng/µl, 20 µl total volume)

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

Further tasks: control sequence

digestion of created pPDV089 for deletion of kanamycin gene

Investigators: Laura, Sabine

Aim:

• inactivation of kanamycin resistence gene

• reason: helper plasmid contains also kanamycin restistence gene

• enable selection of E. coli cells containing both helper plasmid and phage display vector

Time: 14:00-14:30

Reaction components:

• 3 µl pPDV089 (from clones 3F6, 3F8, 2S7 and 2S14)

• 1 µl restriction enzyme NsiI (Fermentas, AG Dittmann)

• 2 µl buffer (Fermentas, AG Dittmann)

• 14 µl water

Reaction conditions:

• over night at 37°C

Digestion of PCR products geneIII and mdnA for cloning into pSB1C3

Investigator: Laura, Sabine

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

Time: 14:30-16:00

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

Investigator: Sabine

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

Time: 16:30-17:30

Material/Method:

• PCR products (mdnA and geneIII)

• 1 % agarose gel

• 1x TAE buffer

• Gel Red

• DNA Ladder Mix (1:10) (Fermentas)

• 6x Loading Dye (Fermentas)

• Gel Red

76th Labday 2011-08-26

miniprep of pUP089 clones

Investigators: Nadine

Time: 2011-08.26, 8:00-9:30

Aim:

• glycerol stocks

• purification of pUP089 plasmids for mdnA library (and screening)

Materials

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

• elution in 50µl Elution buffer

• Check concentration with NanoDrop

• o.n. cultures:

• • pUP089 A I

• • pUP089 A II

• • pUP089 A III

• • pUP089 B I

• • pUP089 B II

• • pUP089 B III
    

Results:

• • pUP089 A I : 498.5 ng/µl

• • pUP089 A II: 465.2 ng/µl

• • pUP089 A III: 462.2 ng/µl

• • pUP089 B I: 426.1 ng/µl

• • pUP089 B II: 443.1 ng/µl

• • pUP089 B III: 448.9 ng/µl

Output:

• # P15: pUP089 A1

• # P16: pUP089 A II

• # P17: pUP089 A III

• # P18: pUP089 B I

• # P19: pUP089 B II

• # P20: pUP089 B III

• glycerol stocks in XL1 blue:

• • # G2 pUP089 A I

• • # G3 pUP089 A II

• • # G4 pUP089 A III

• • # G5 pUP089 B I

• • # G6 pUP089 B II

• • # G7 pUP089 B III

PCR of complete mdn cluster for biobrick-construction

Investigators: Steffi, Nadine, Katharina

Material:

• pARW089

• pARW071

• dNTPs

• primer 77, 78, 79, 80

• HF Phusion Buffer 5x* Phusion Polymerase

Method:

• 2µl pARW089, 1µl pARW071, respectively

• 1µl dNTPs

• 2µl forward primer (10mM)

• 2µl reverse primer (10mM)

• 10µl HF Phusion Buffer 5x

• 0.5 µl Phusion Polymerase

• 31.5 µl water (total volume= 50µl)

• reaction

Step	Temperature	Time
Hot Start	95°C	Hold
Initial denaturation	95°C	30 sec
Denaturation	95°C	30 s
Annealing	51°C	60 s
Extension	68°C	6.5 min
DenaturationII	95°C	30 s
AnnealingII	63°C	60 s
ExtensionII	68°C	6.5 min
Final extension	68°C	50 min

Result:

• three different PCR samples

• • PCR sample 1: pARW071 + primer 77/78 = mdnABCDE71

• • PCR sample 2: pARW089 + primer 77/79 = mdnABCDE89+T7

• • PCR sample 3: pARW089 + primer 77/80 = mdnABCDE89

Further Tasks:

• gel electrophoresis

• PCR clean up

• restriction enzyme digestion

• ligation

• transformation

digest of PCR products from 25.08.2011

Investigators: Niels

Aim:

• digest of PCR - products

Materials: PCR from 25.08.2011

• PCR loop

• PCR 1 Libary

• PCR 2 Libary

Sample:

• 0,5 µl DNA

• 1 µl Aat II (Roche)

• 3 µl 10x Buffer (Roche)

• 25,5 µl water

• total volume 30 µl

• • 37°C

• • 3 hours

further tasks:

• ligation with pUP089

Repeated ligation of digested mdn fragments and pSB1C3 ( from 2011-08-24)

Investigators: Jessica, Nadja

Time: 2011-08-26, 18:00

Materials

Ligation:

Insert	[µl]	Vector	[µl]
mdnB	6	C3	2
mdnC	6	C3	2
mdnD	7	C3	1
mdnE	5	C3	3
mdnBC	5	C3	3
mdnDE	5	C3	3
mdnABC	5	C3	3
H20	5	C3	3

• 1 µl 10x Buffer

• 1 µl T4 Ligase

• over night by 14°C
  

Further tasks:

• transformation

Agarose gel and Gel extraction of pUP089 and the three libraries as well as their ligation

Investigators: Jessica, Nadine, Nadja

Time: 2011-08-26, 16:00

Aim

• Build up a Library of mutated mdnA`s
  

Materials

• digests of:

• pUP089

• libraries 800, 2000, loop
  

Agarose gel

Gel 1

• 1%: 0.5 g agarose + 50 ml 1x TAE, adding 2 µl gel red

lane	Sample	Volume in µl	Expected size in bp
M	GeneRuler™ DNA Ladder Mix (diluted 1:10)	12
1	-
2	digest pUP089	36	10029, 161

Gel 2

• 2%: 1 g agarose + 50 ml 1x TAE, adding 2 µl gel red

lane	Sample	Volume in µl	Expected size in bp
M	GeneRuler™ 100bp DNA Ladder	2
1
2	Library 1	36	161
3
4	Library 2	36	161
5	-
6	Library Loop	36	161

Gel extraction

• Promega- Wizard SV Gel and PCR Clean Up System

• Eluation with 50µl Nuclease- Free Water
  

Nano- Drop :Concentrations

• Library 1- digested- purified 11-26: 6,2 ng/µl

• Library 2- digested- purified 11-26: 6,1 ng/µl

• Library Loop- digested- purified 11-26: 7,5 ng/µl

• pUP089: 11,3 ng/µl
  

Dephosphorylation of pUP089

• add 5µl Multicore 10x Buffer (Promega)

• add 1µl Thermosensitive Alkaline Phosphatase (Promega)

• incubate at 37°C for 15 min

• heat inactivation at 74°C for 15 min
  

Ligation

Insert	[µl]	Vector	[µl]
Library 1	1	pUP089	7
Library 2	1	pUP089	7
Library Loop	1	pUP089	7
H2O	1	pUP089	7

• 1 µl 10x Buffer

• 1 µl T4 Ligase

• over night at 14°C
  

Further tasks:

• transformation

over night cultures of XL1 blue pSB1C3+mdn-combination

Time: 2011-08-26, 10:00-12:00

Investigators: Nadine, Nadja

Material:

• plates from 2011-08-25, Niels

• • pSB1C3+mdnB clone 1

• • pSB1C3+mdnB clone 2

• • pSB1C3+mdnC clone 1

• • pSB1C3+mdnCclone 2

• • pSB1C3+mdnD clone 1

• • pSB1C3+mdnD clone 2

• • pSB1C3+mdnE clone 1

• • pSB1C3+mdnE clone 2

• • pSB1C3+mdnABC clone 1

• • pSB1C3+mdnABC clone 2

• • pSB1C3+mdnBC clone 1

• • pSB1C3+mdnBC clone 2

• • pSB1C3+mdnDE clone 1

• • pSB1C3+mdnDE clone 2

• 25 mg/ml chloramphenicol

• LB-medium

Protocol:

• 5 ml LB and 5 µl chloramphenicol

• tip w/ colony

• incubation: 37°C, 200 rpm, over night

Glycerol stocks of E. coli containing created pPDV089

Investigator: Sabine

Time: 10:30-10:30

Material/Method:

• 300 µl glycerol per stock

• 700 µl cell culture per stock

• storage at 80 °C

Gel electrophoresis to control digestion of pPDV089 with NsiI

Investigators: Sabine

Time: 10:30-12:00

Aim: control of digestion and purification

Material/Method:

• digested pPDV089 (clones 3F6, 3F8, 2S7 and 2S14)

• 1 % agarose gel

• 1x TAE buffer

• Gel Red

• DNA Ladder Mix (1:10) (Fermentas)

• 6x Loading Dye (Fermentas)

• Gel Red

• purification see protocol NucleoSpin ExtractIII kit

Result:

• right band sizes

• vector ca. 10 kb

• 2 bands closed to each other near 10 kb (except 3F8), both were separately cut out of the gel)

• deleted part of kanamycin gene ca. 300 bp

Further tasks:

• purification

• re-ligation

PCR of geneIII

Investigators: Sabine

Time: 12:00-14:00

Aim: amplification of geneIII for cloning into pARW089

Reaction Components:

• 2 µl/ 20 ng geneIII (cut out from pARW089)

• 0,25 µl Taq Polymerase S (BioScience)

• 1 µl dNTPs

• 1 µl primer pf_geneIII_EheI_XbaI_NgoMIV

• 1 µl primer pr_geneIII_iGEM_AatII

• 5 µl 10x PCR Buffer S

• 39,75 µl DNase free water

• prufication see protocol NucleoSpin ExtractII kit

Further tasks:

• digestion

Ligation go geneIII and mdnA into pSB1C3

Investigator: Sabine

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

Time: 14:00-15:30

Material/Method:

• 1,9 µl PCR product geneIII (36 µg/µl)

• 6,1 µl pSB1C3 (17 ng/µl)

• 1 µl T4 ligase buffer (Fermentas)

• 1 µl T4 Ligase (Fermentas)

• 1 h at room temperature

• 2,4 µl PCR product mdnA (12 µg/µl)

• 6,6 µl pSB1C3 (17 ng/µl)

• 1 µl T4 ligase buffer (Fermentas)

• 1 µl T4 Ligase (Fermentas)

• 1 h at room temperature

• 1,9 µl PCR product mdnA (12 µg/µl)

• 1,6 µl PCR product geneIII (36 µg/µl)

• 4,5 µl pSB1C3 (17 ng/µl)

• 1 µl T4 ligase buffer (Fermentas)

• 1 µl T4 Ligase (Fermentas)

• 1 h at room temperature

Further task: transformation

Re-ligation of NsiI-digested pPDVs089

Investigator: Sabine

Aim: create pPDV089 without kanamycin resistence

Time: 14:30-16:00

Material/Method:

• 17 µl NsiI-digested pPDVs089 (3F6, 3F8, 2S7 and 2S14, DNA from upper and lower bands)

• 1 µl T4 ligase buffer (Fermentas)

• 2 µl T4 Ligase (Fermentas)

• 1 h at room temperature

Further task: transformation

Ligation of geneIII into pARW089

Investigator: Sabine

Aim: cloning of geneIII into pARW089

Time: 15:00-17:30

Material/Method:

• 1,5 µl PCR product geneIII (17 µg/µl)

• 6,5 µl pARW089 (30 ng/µl)

• 1 µl T4 ligase buffer (Fermentas)

• 1 µl T4 Ligase (Fermentas)

• 1 h at room temperature

Further task: transformation

Transformation of created vectors in E. coli

Investigators: Sabine

Aim:amplification of vectors

Time: 18:00-21:00

Material:

• pPDV089 without kanamycin resistence (3F6, 3F8, 2S7 and 2S14)

• pARW089 containing only geneIII (no mdnA)

• pSB1C3 containing mdnA, geneIII or mdnA/geneIII

• agar plates containing chloramphenicol (pSB1C3)

• agar plates containing kanamycin, ampicillin and 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

77th Labday 2011-08-27

Agarose gel of PCR prducts mdnABC and PCR purification

Time: 2011-08-27, 10:00-12:00

Investigators: Nadine

Aim:

• prove PCR

• purification of PCR prducts
  

Materials:

• PCR products from 2011-08-26 , Katharina

• • mdnABCDE71

• • mdnABCDE89+T7

• • mdnABCDE89

• Promega- Wizard SV Gel and PCR Clean Up System (elution in 50 µl elution buffer)

• Agarose (BioRad)

• 1x TAE

• gel red

• loading dye (promega)

• 1:100 ladder mix (Fermentas)

Protocol:

• 1%: 0.5 ge agaros + 50 ml 1xTAE buffer, 2 µl gel red

• PCR clean up: following protocol from kit

Results:

lane	Sample	Volume in µl	Expected size in bp
M	Ladder	12
1
2	mdnABCDE from pARW071	12 (2 µl PCR product)	~6500
3
4	mdnABCDE from pARW089 w/ T7	12 (2 µl PCR product)	~6500
5	-
6	mdnABCDE from pARW089 without T7	12 (2 µl PCR product)	~6500

• no product for mdnABCDE of pARW089 w/ T7

• PCR clean up:

• • mdnABCDE from pARW071: 36.6 ng/µl

• • mdnABCDE from pARW089 w/ T7: 37.6 ng/µl

• • mdnABCDE from pARW089 without T7: 63.9 ng/µl

Further tasks:

• digest w/ EcoRI and SpeI (also pSB1C3)

• repeat PCR (third time)

competent cells - E.coli XL1 blue & RF308

Investigator: 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 68 Eppis (1,5µl)

• get liquid nitrogen

• prepare 5 ml LB-Medium with the specific antibiotic (for XL1-blue: Tet, for BL21: none!), 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: 50µl per tube and store immediately at liquid nitrogen and afterwards at -80 °C

Results:

• 34 tubes RV308 for expression(50 µl competent cells at -80°C)

• 34 tubes XL1 blue for transformation(50 µl competent cells at -80°C)

Further tasks:
check by transformation

miniprep of several mdn clones and glycerole stocks

Investigators: Nadine, Katharina

Time: 2011-08-27, 11:00-12:00

Aim:

• glycerol stocks

• purification of pSB1C3+mdnX plasmids for mdnX biobricks (and screening)

Materials

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

• elution in 50µl Elution buffer

• Check concentration with NanoDrop

• o.n. cultures (from 2011-08-26, Nad/Nad):

• • pSB1C3+mdnB clone 1

• • pSB1C3+mdnB clone 2

• • pSB1C3+mdnC clone 1

• • pSB1C3+mdnCclone 2

• • pSB1C3+mdnD clone 1

• • pSB1C3+mdnD clone 2

• • pSB1C3+mdnE clone 1

• • pSB1C3+mdnE clone 2

• • pSB1C3+mdnABC clone 1

• • pSB1C3+mdnABC clone 2

• • pSB1C3+mdnBC clone 1

• • pSB1C3+mdnBC clone 2

• • pSB1C3+mdnDE clone 1

• • pSB1C3+mdnDE clone 2

Results:

• • pSB1C3+mdnB clone 1: 778.3 ng/µl

• • pSB1C3+mdnB clone 2: 600.5 ng/µl

• • pSB1C3+mdnC clone 1: 1031.7 ng/µl

• • pSB1C3+mdnCclone 2: 843.3 ng/µl

• • pSB1C3+mdnD clone 1: 482.8 ng/µl

• • pSB1C3+mdnD clone 2: 541.1 ng/µl

• • pSB1C3+mdnE clone 1: 734.4 ng/µl

• • pSB1C3+mdnE clone 2: 768.2 ng/µl

• • pSB1C3+mdnABC clone 1: 853.4 ng/µl

• • pSB1C3+mdnABC clone 2: 690.3 ng/µl

• • pSB1C3+mdnBC clone 1: 626.9 ng/µl

• • pSB1C3+mdnBC clone 2: 764 ng/µl

• • pSB1C3+mdnDE clone 1: 899.4 ng/µl

• • pSB1C3+mdnDE clone 2: 525.6 ng/µl

Output:

• plasmids: see results (stored in -20°C: green box, red label: mdn biobricks)

• glycerol stocks in XL1 blue (stored in -80°C, glycerole box #2):

• • pSB1C3+mdnB clone 1, 27.8.11, Nad/Kat

• • pSB1C3+mdnB clone 2, 27.8.11, Nad/Kat

• • pSB1C3+mdnC clone 1, 27.8.11, Nad/Kat

• • pSB1C3+mdnCclone 2, 27.8.11, Nad/Kat

• • pSB1C3+mdnD clone 1, 27.8.11, Nad/Kat

• • pSB1C3+mdnD clone 2, 27.8.11, Nad/Kat

• • pSB1C3+mdnE clone 1, 27.8.11, Nad/Kat

• • pSB1C3+mdnE clone 2, 27.8.11, Nad/Kat

• • pSB1C3+mdnABC clone 1, 27.8.11, Nad/Kat

• • pSB1C3+mdnABC clone 2, 27.8.11, Nad/Kat

• • pSB1C3+mdnBC clone 1, 27.8.11, Nad/Kat

• • pSB1C3+mdnBC clone 2, 27.8.11, Nad/Kat

• • pSB1C3+mdnDE clone 1, 27.8.11, Nad/Kat

• • pSB1C3+mdnDE clone 2, 27.8.11, Nad/Kat

Further Tasks:

• test digest (today)

• sequencing (Monday, 2011-08-29)

Digestion of PCR products of complete mdn-cluster (mdnABCDE)

Investigators: Nadine, Katharina

Materials

• pSB1C3

• purified PCR products of mdn-cluster

• NEB Buffer 4

• EcoRI

• SpeI

• BSA
  

Method

• digestion of inserts (PCR products of mdn-cluster)

• • 50 µl purified PCR product

• • 1.5 µl EcoRI

• • 1.5 µl SpeI

• • 6 µl NEB Buffer 4

• • 0.6 µl BSA

• • 0.4 µl sterile water

• digestion of vector (pSB1C3 235.0 ng/µl)

• • 3 µl NEB Buffer 4

• • 4 µl pSB1C3

• • 0.3 µl BSA

• • 1.5 µl EcoRI

• • 1.5 µl SpeI

• • 19.7 µl sterile water

Agarose gel

• 0.8%: 0.4 g agarose + 50 ml 1x TAE, adding 2 µl gel red

lane	Sample	Volume in µl	Expected size in bp
M	Ladder	12
1	mdnABC from pARW071, digested	??	~6500
2	mdnABC from pARW089 w/ T7, digested	??	~6500
3	mdnABC from pARW089 without T7, digested	??	~6500
4	pSB1C3, digested		~150, 2400

Conclusions

• only mdnABCDE89 has worked

Further tasks:

• repeat PCR for mdnABCDE71 and mdnABCDE89 and mdnABCDE89+T7

• Transformation of pSB1C3+mdnABCDE89 in XL1 blue

Transformation of ligation: pUP089 + Lib-1/2, pSB1c3 + several mdn

Investigator: Niels

Material:

• XL1-blue (competent)

• ligation products from ligation from 2011-08-26/25

old competent cells

• • pSB1C3 + H₂O

• • pSB1C3 + mdnBC

• • pSB1C3 + mdnB

• • pUP089 + puB control

• • pUP089 + Lib-loop

• • pUP089 + Lib-1

• • pUP089 + Lib-2

new competent cells

• • pSB1C3 + mdnE

• • pSB1C3 + H₂O

• • pSB1C3 + mdnD

• • pSB1C3 + mdnABC

• • pSB1C3 + mdnDE

• • pSB1C3 + mdnC

• • puP089 + Lib-1

• LB-Agar

• • chloramphenicol

• • kanamycine

Method:

• addition of 2 µl ligation reaction to XL1-blue cells
  

• incubation 60 min on ice,
  

• heat shock 90 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:

• • pUP089

• • • 100 µg/ml kanamycin
      

• • pSB1C3

• • • 100 µg/ml chloramphenicol

• incubation for 20 hrs at 37°C
  

Further tasks:
control cell clones

Ligation of 3x pUP089 + Lib-2

Investigators: Niels

Material:

• pUP089 (restricted with EheI/AatII) (Vector)

• Lib-2 (Insert)

Method:

• 7 µl pUP089

• 1 µl Insert

• 1 µl Ligase buffer

• 1 µl T4 Ligase

Further tasks:

Transformation

Ligation of mdnABCDE89 + pSB1C3

Investigators: Katharina

Material:

• pSB1C3 (Vector)

• mdnABCDE89 (Insert)

Method:

• ligation

• • 7 µl purified PCR product of mdn-cluster

• • 1 µl pSB1C3

• • 1 µl Ligase buffer

• • 1 µl T4 Ligase

• ligation control

• • 7 µl sterile water

• • 1 µl pSB1C3

• • 1 µl Ligase buffer

• • 1 µl T4 Ligase

Mini-Prep of the positiv clones from an overnight culture

Investigator: Stefan

Material:

• Mini-Prep Kit (machery & Nagel)
  

Method:

Standard protocol 5.1 for plasmid preparation. And do glycerol stock cultures of all them.

Further tasks:

Send plasmids for sequencing

test digest of pSB1C3+mdncombinations (several mdn clones/combinations)

Time: 2011-08-27,16:30-18:30

Investigators: Nadine

Aim: prove of Insert (mdn combinations)

Materials:

• pSB1C3+mdn-combinations (miniprep from today, see above, Nad/Kat)

• HindIII, PvuII, ClaI, AvaI, HpaI (NEB)

• Buffer 4 and 2 (NEB, 10x)

• BSA

Plan:

insert	plasmid size in bp	enzymes	buffer	Expected size in bp
mdnC	3385	HindIII	2	363, 3022
mdnD	2944	PvuII	4	220, 591, 2133
mdnE	4474	ClaI	4 + BSA	769, 3705
mdnABC	4852	HindIII, AvaI	2	363, 836, 3653
mdnBC	4419	HindIII, AvaI	2	363, 836, 3220
mdnDE	5154	HpaI	4	1329, 3825

Digestion protocol (one enzyme)

• 1 µl DNA

• 0.5 µl enzyme (see table)

• 2 µl 10x buffer

• 16.5 µl pure water

Digestion protocol (two enzymes)

• 1 µl DNA

• 0.5 µl enzyme I

• 0.5 µl enzyme II

• 2 µl 10x buffer

• 16 µl pure water

Digestion protocol (two enzymes and BSA)

• 1 µl DNA

• 0.5 µl enzyme I

• 0.5 µl enzyme II

• 2 µl 10x buffer

• 0.2 µl BSA

• 15.8 µl pure water

• total: 20µl

• 37°C for over night

Conclusions:

• annotation of mdnB in pARW089 (genious) might be wrong

Further tasks:

• agarose gel

• check annotation of mdnB

Troubleshooting of transformed cells

Investigator: Stefan

Aim:

• check of vector is digested at all
  

Materials:

heat shock transformation

Method:

heat shock transformation

Output:

78th Labday 2011-08-28

comparison of "new" and "old" competent cells

Investigators: Katharina

Results:

check plates from transformation (pSB1C3 w/ mdn combinations in XL1 blue, 2011-08-27)

Time: 2011-08-28, 12:00-12:30

Investigators: Katharina, Nadine

Aim:

• build biobricks w/ mdn combinations

Material:

• plates

• scanner

Results:

conclusions:

• Trafo has worked

Further tasks:

• pick colonies from plates

• mini prep tomorrow

• test digest (see 2011-08-27)

• sequencing

Agarose gel of PCR prducts mdnABC and PCR purification

Time: 2011-08-27, 10:00-12:00

Investigators: Nadine, Katharina

Aim:

• prove insert of pSB1C3 with mdn-combinations (biobricks) (miniprep from 2011-08-27, Nad/Kat)

Materials:

• Agarose (BioRad)

• 1x TAE

• gel red

• loading dye (promega)

• 1:100 ladder mix (Fermentas)

• 1kb DNA ladder (Promega)

Protocol:

• 2 x 1%: 0.5 g agarose + 50 ml 1xTAE buffer, 2 µl gel red

Results:

• gel 1

lane	sample	enzymes	volume in µl	Expected size in bp
M1	1:100 DNA ladder mix, Fermentas		12
1	-	-	-	-
2	mdnABC 1	HindIII, AvaI	24	363, 836, 3653
3	mdnABC 2	HindIII, AvaI	24	363, 836, 3653
4	-	-	-	-
5	mdnBC 1	HindIII, AvaI	24	363, 836, 3220
6	mdnBC 2	HindIII, AvaI	24	363, 836, 3220
7	-	-	-	-
8	mdnC 1	HindIII	24	363, 3022
9	mdnC 2	HindIII	24	363, 3022
10	-	-	-	-
M2	1kb DNA ladder, Promega	-	6	-

• gel 2

lane	sample	enzymes	volume in µl	Expected size in bp
M1	1:100 DNA ladder mix, Fermentas		12
1	-	-	-	-
2	mdnD 1	PvuI	24	220, 591, 2133
3	mdnD 2	PvuI	24	220, 591, 2133
4	-	-	-	-
5	mdnDE 1	HpaI	24	1329, 3825
6	mdnDE 2	HpaI	24	1329, 3825
7	-	-	-	-
8	mdnE 1	ClaI	24	769, 3705
9	mdnE 2	ClaI	24	769, 3705
10	-	-	-	-
M2	1kb DNA ladder, Promega	-	6	-

Conclusions:

• only D1 (Gel 2, lane 2) seems to be okay

over night cultures of XL1 blue pSB1C3+mdn-combination

Time: 2011-08-26, 10:00-12:00

Investigators: Katharina

Material:

• plates from 2011-08-27, Niels

new competent cells

• • pSB1C3 + mdnE

• • pSB1C3 + mdnD

• • pSB1C3 + mdnABC

• • pSB1C3 + mdnDE

• • pSB1C3 + mdnC

• each plate: 3 clones

• 25 mg/ml chloramphenicol

• LB-medium

Protocol:

• 5 ml LB and 5 µl chloramphenicol

• tip w/ colony

• incubation: 37°C, 200 rpm, over night

Output:

• 15 tubes in shaker:

• • pSB1C3 + mdnE: 1, 2, 3

• • pSB1C3 + mdnD: 1, 2, 3

• • pSB1C3 + mdnABC: 1, 2, 3

• • pSB1C3 + mdnDE: 1, 2, 3

• • pSB1C3 + mdnC: 1, 2, 3

Further tasks:

• mini prep tomorrow

Production of TEV and 14_3C biobricks part 1

Aim: indtroduction of iGEM restriction sites to produced mutated TEV and 14_3C Fragments.

Primer TEV:

(1) f_TEV_ACCAGC, r_TEV_iGEM_Eco81l

(2) r_TEV_ACCAGC, f_TEV_iGEM

Primer 14_3C:

(1) f_14_3C_ACCAGC, r_14_3C_iGEM_Eco81l

(2) r_14_3C_ACCAGC, f_14_3C_iGEM

Methode:

PCR

• Template: 1µl (TEV or 14_3C <10ng)

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

• 5µl 10x Amplification buffer S
  

• 2µl 25mM MgCl2
  

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

• 35,5µl of pure water
  

• 0,5µl TaqPol
  

Program:

• Denat: 3min 94°C

• 5x:

Denat: 30sec 94°C

Anneal:30sec 55°C

Extend:30sec 72°C

• 25x:

Denat: 30sec 55°C

Anneal:30sec 55°C

Extend:30sec 72°C

• Final Extend: 10min 72°C

Further Tasks:

PCR purification of PCR products and Annealing of produced fragments

Transformation of pSB1C3+mdnABCDE89 and pUP089+library2

Investigators: Katharina, Steffi

Aim: Transformation of Ligation

Time: 2011-08-28, 11:00-13:00

Materials:

• competent E. coli cells (XL1-Blue, 2011-08-27, Steffi)

• ligation products: pSB1C3+mdnABCDE89 (2011-08-27, Katharina), pUP089+Lib2 (2011-08-27, Niels)

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 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 (Cm, Kan)

• storage over night at 37°C

Further tasks:

• Picking clones for overnight culture

• Producing glycerol stocks

Output:

• 10 plates w/ Kan: mdn-lib1

• 2 plates w/ Cm: pSB1C3+mdnABCDE89 (and control)

overnight culture of picked E. coli clones transformed with pPDV089 to control deletion of kanamycin gene

Investigators: Sabine

Aim: control deletion of kanamycin gene in cteated pPDV089

Method/Materials:

• 3 clones from pPDV089_3F6

• 3 clones from pPDV089_3F8

• 3 clones from pPDV089_2S7

• 3 clones from pPDV089_2S14

• each clone picked 2x: one for growing in LB medium with kanamycin, one in LB medium with ampicillin

• 5 ml LB medium per clone

• storage over night at 37°C and 800 rpm
  

Further tasks:

• control, whether clones grew in presence of ampicillin but not of kanamycin
  

Results:

• clones grew in presence of both kanamycin and ampicillin

• kanamycin deletion didn`t work out

79th Labday 2011-08-29

check transformation from yesterday (pSB1C3+mdnABCDE89 and pUP089+mdn-Lib2)

Investigators: Nadine, Steffi

Aim: check transformation

Time: 2011-08-28, 8:20-8:40

Materials:

• Transformations from yesterday (2011-08-28, Kat)

Conclusions:

• transfomations didn´t work

• repeat PCRs

Further tasks:

• repeat PCR for mdnABCDE

• repeat PCR for mdnA-Library

miniprep of several mdn clones

Investigators: Steffi

Time: 2011-08-29, 07:00-09:30

Aim:

• purification of 15x pSB1C3+mdnX plasmids for mdnX biobricks (and screening) from over night cultures (Katharina, 2011-08-28)

Materials

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

• elution in 50µl Elution buffer

• Check concentration with NanoDrop

• o.n. cultures (from 2011-08-28, Kat):

• • pSB1C3+mdnC clone 1

• • pSB1C3+mdnC clone 2

• • pSB1C3+mdnC clone 3

• • pSB1C3+mdnD clone 1

• • pSB1C3+mdnD clone 2

• • pSB1C3+mdnD clone 3

• • pSB1C3+mdnE clone 1

• • pSB1C3+mdnE clone 2

• • pSB1C3+mdnE clone 3

• • pSB1C3+mdnABC clone 1

• • pSB1C3+mdnABC clone 2

• • pSB1C3+mdnABC clone 3

• • pSB1C3+mdnDE clone 1

• • pSB1C3+mdnDE clone 2

• • pSB1C3+mdnDE clone 3

Results:

• • pSB1C3+mdnC clone 1: 434,1 ng/µl

• • pSB1C3+mdnC clone 2: 304,4 ng/µl

• • pSB1C3+mdnC clone 3: 620,7 ng/µl

• • pSB1C3+mdnD clone 1: 282,6 ng/µl

• • pSB1C3+mdnD clone 2: 413,4 ng/µl

• • pSB1C3+mdnD clone 3: 440,0 ng/µl

• • pSB1C3+mdnE clone 1: 543,7 ng/µl

• • pSB1C3+mdnE clone 2: 573,5 ng/µl

• • pSB1C3+mdnE clone 3: 440,5 ng/µl

• • pSB1C3+mdnABC clone 1: 542,3 ng/µl

• • pSB1C3+mdnABC clone 2: 267,5 ng/µl

• • pSB1C3+mdnABC clone 3: 263,6 ng/µl

• • pSB1C3+mdnDE clone 1: 366,5 ng/µl

• • pSB1C3+mdnDE clone 2: 440,2 ng/µl

• • pSB1C3+mdnDE clone 3: 340 ng/µl

Output:

• plasmids: see results (stored in -20°C: green box, red label: mdn biobricks)

Further Tasks:

• test digest (today)

• sequencing (today?)

test digest of pSB1C3+mdncombinations (several mdn clones/combinations)

Investigators: Steffi

Aim: prove of Insert (mdn combinations)

Materials:

• pSB1C3+mdn-combinations (miniprep from today, see above, Steffi)

• HindIII, PvuII, ClaI, AvaI, HpaI (NEB)

• Buffer 4 and 2 (NEB, 10x)

• BSA

Plan:

insert	plasmid size in bp	enzymes	buffer	Expected size in bp
mdnC	3385	HindIII	2	363, 3022
mdnD	2944	PvuII	4	220, 591, 2133
mdnE	4474	ClaI	4 + BSA	769, 3705
mdnABC	4852	HindIII, AvaI	2	363, 836, 3653
mdnDE	5154	HpaI	4	1329, 3825

Digestion protocol (one enzyme)

• 1 µl DNA

• 0.5 µl enzyme (see table)

• 2 µl 10x buffer

• 16.5 µl pure water

Digestion protocol (two enzymes)

• 1 µl DNA

• 0.5 µl enzyme I

• 0.5 µl enzyme II

• 2 µl 10x buffer

• 16 µl pure water

Digestion protocol (two enzymes and BSA)

• 1 µl DNA

• 0.5 µl enzyme I

• 0.5 µl enzyme II

• 2 µl 10x buffer

• 0.2 µl BSA

• 15.8 µl pure water

• total: 20µl

• 37°C for 1,5 h

Result gel:

lane	sample	enzymes	volume in µl	Expected size in bp
M1	1:100 DNA ladder mix, Fermentas		12
1	-	-	-	-
2	mdnC 1	HindIII	24	363, 3022
3	mdnC 2	HindIII	24	363, 3022
4	mdnC 3	HindIII	24	363, 3022
5	mdnD 1	PvuII	24	220, 591, 2133
6	mdnD 2	PvuII	24	220, 591, 2133
7	mdnD 3	PvuII	24	220, 591, 2133
8	mdnE 1	ClaI	24	769, 3705
9	mdnE 2	ClaI	24	769, 3705
10	mdnE 3	ClaI	24	769, 3705
11	mdnDE 1	HpaI	24	1329, 3825
12	-	-	-
13	mdnDE 2	HpaI	24	1329, 3825
14	mdnDE 3	HpaI	24	1329, 3825
15	mdnABC 1	HindIII, AvaI	24	363, 836, 3653
16	mdnABC 2	HindIII, AvaI	24	363, 836, 3653
17	mdnABC 3	HindIII, AvaI	24	363, 836, 3653

Further tasks:

• sequencing of mdnC (2+3), mdnD (1+3), mdnE (2)

• repeat PCR of mdnB (wait for new primer), mdnABC and mdnDE

competent cells - E.coli XL1 blue

Investigator: 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 (1,5µl)

• get liquid nitrogen

• prepare 5 ml LB-Medium with the specific antibiotic (for XL1-blue: 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: 50µl per tube and store immediately at liquid nitrogen and afterwards at -80 °C

Results:

• 80 tubes XL1 blue 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 XL1 blue

Investigator: Katharina, Steffi

Aim: check resistance of competent XL1 blue-cells on agar plates with different antibiotics

Materials/Methods:

• • competent XL1 blue-cells from 2011-08-27 and 2011-08-29 (Steffi)

• • LB-plates with Tetracycline, Chloramphenicol, Kanamycine, Ampicillin

• • plating 50 µl on agar plates

• • incubate at 37°C over night

Further tasks:
control agar plates

Oligo-Fillin for mdnA-Library (repetition)

Time: 2011-08-29, 8:30-11:30

Investigators: Nadine

Materials

• Primer, 25 µM :

• • # 76

• • # 74: o_foc_library_2

• • # 72: o_loop_con_switch

• Klenow-Buffer 10X

• Klenow Fragment

• dNTPs

• water

• Agarose (BioRad)

• 1x TAE

• gel red

• loading dye (promega)

• 100bp DNA ladder (Fermentas)

• 1kb DNA ladder (Promega)

Protocol:

• Reaction mix

• • 1 µl fw-Oligonucleotide (#76)

• • 1 µl rev-Oligonucleotide (#74 or #72)

• • 2 µl dNTPs (10 mM)

• • 2 µl Klenow-Fragment

• • 14 µl water

• • total volume: 20 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:

1.5 %: 0.75 g in 50 ml TAE

Results:

• Output:

• • • mdnA-Lib2, Nad, 29.8.11 (~170bp)

• • • mdnA-loop, Nad, 29.8.11 (~170bp)

Further tasks:

• PCR purification

sequencing of TEV and 14_3C clones

Aim: get sequences

Methode:

send samples to GATC

Further Tasks:

Restriction of pUP089 (vector) and Lib-2/loop(Insert/PCR)

Investigator: Niels

Aim: generate vector and insert for ligation and transformation to generate a Libary

Materials/Methods:

• pub089 (A)

• • 5 µl DNA

• • 0,5 µl Ehe I

• • 0,5 µl Aat II

• • 3 µl 10x Buffer

• • 21 µl water

• • • total volume: 30µl

• pub089 (B)

• • 1 µl DNA

• • 0,5 µl Ehe I

• • 0,5 µl Aat II

• • 3 µl 10x Buffer

• • 25 µl water

• • • total volume: 30µl

• Lib-2 (A)

• • 20 µl DNA

• • 0,5 µl Aat II

• • 3 µl 10x Buffer

• • 6,5 µl water

• • • total volume: 30µl

• Lib-2 (B)

• • 1 µl DNA

• • 0,5 µl Aat II

• • 3 µl 10x Buffer

• • 25,5 µl water

• • • total volume: 30µl

• Loop

• • 1 µl DNA

• • 0,5 µl Aat II

• • 3 µl 10x Buffer

• • 25,5 µl water

• • • total volume: 30µl

over night

Further tasks:

• purification

• dephosphorylation

• ligation

• transformation

PCR of complete mdn cluster for biobrick-construction

Investigators: Niels, Nadine

Material:

• pARW089

• pARW071

• dNTPs

• primer 77, 78, 79, 80

• HF Phusion Buffer 5x* Phusion Polymerase

Method:

• 2µl pARW089, 1µl pARW071, respectively

• 1µl dNTPs

• 2µl forward primer (10mM)

• 2µl reverse primer (10mM)

• 10µl HF Phusion Buffer 5x

• 0.5 µl Phusion Polymerase

• ad 50 µl water

• reaction

Step	Temperature	Time
Hot Start	98°C	Hold
Initial denaturation	98°C	30 sec
Denaturation	95°C	30 s
Annealing	59°C	60 s
Extension	72°C	3,5 min
DenaturationII	95°C	30 s
AnnealingII	59°C	60 s
ExtensionII	72°C	3.5 min
Final extension	68°C	5 min

Result:

• three different PCR samples

• • PCR sample 1: pARW071 + primer 77/78 = mdnABCDE71

• • PCR sample 2: pARW089 + primer 77/79 = mdnABCDE89+T7

• • PCR sample 3: pARW089 + primer 77/80 = mdnABCDE89

Further Tasks:

• gel electrophoresis

• PCR clean up

• restriction enzyme digestion

• ligation

• transformation

Alignment of sequenced pPDV089 and model of pPDV089

Investigator: Sabine

Aim: control of generated phage display vector pPDV089

Materials/Methods:

• file sent from GATC

• software Genious

Results:

• 4 positive clones, pPDV089 containing mdnA and geneIII (3F6, 3F8, 2S7, 2S14)

Further task:

• deletion of kanamycin gene

PCR of geneIII and mdnA

Investigators: Sabine

Aim:

• amplification of mdnA and geneIII for cloning into pSB1C3

• amplification of geneIII for cloning into pARW089

Reaction Components:

• 2 µl/ 20 ng geneIII (cut out from pARW089

• 0,25 µl Taq Polymerase S (BioScience)

• 1 µl dNTPs

• 1 µl primer pf_geneIII_EheI_XbaI_NgoMIV

• 1 µl primer pr_geneIII_iGEM_AatII

• 5 µl 10x PCR Buffer S

• 39,75 µl water

• 5 µl/ 50 ng pak bla KDIR (mdnA)

• 0,25 µl Taq Polymerase S (BioScience)

• 1 µl dNTPs

• 1 µl primer pf_mdnA_iGEM_EheI

• 1 µl primer pr_mdnA_iGEM_AatII

• 5 µl 10x PCR Buffer S

• 36,75 µl water

Further tasks:

• analytic gel electrophoresis

• purification

• digestion

80th Labday 2011-08-30

Agarose gel of PCR of complete mdn cluster for biobrick-construction

Time: 2011-08-27, 08:00-09:00

Investigators: Nadine, Steffi

Aim:

• prove insert of pARW071/ pARW089 with complete mdn-cluster (PCR-product from 2011-08-29, Nadine)

Materials:

• Agarose (BioRad)

• 1x TAE

• gel red

• loading dye (promega)

• 1kb DNA ladder (Promega)

Protocol:

• 1 x 1%: 0.5 g agarose + 50 ml 1xTAE buffer, 2 µl gel red

Results:

• gel

lane	sample	volume in µl	Expected size in bp
M1	1kb DNA ladder (Promega)	10	-
1	mdnABCDE71	2
2	mdnABCDE89	2
3	mdnABCDE89+T7	2

Conclusions:

• PCR didn`t work

Further Tasks:

• repeat PCR

• gel electrophoresis

• PCR clean up

• restriction enzyme digestion

• ligation

• transformation

Repeat PCR of complete mdn cluster for biobrick-construction

Investigators: Nadine, Vanessa, Steffi

Aim: repeat PCR of complete mdn cluster for biobrick-construction from 2011-08-29

Material:

• pARW089

• pARW071

• dNTPs

• primer 77, 78, 79, 80

• Bio-X-ACT Long DNA Polymerase (Bioline)

Method:

• 1µl pARW089 (~ 3.3ng) , 1µl pARW071 (~ 5ng)

• 0.5µl dNTPs (100 mM)

• MgCl2 (50 mM)

• 1.5µl forward primer (100µM)

• 1.5µl reverse primer (100µM)

• 5µl OptiBuffer 10x

• 1µl Bio-X-ACT Long DNA Polymerase (Bioline)

• add 50 µl water

• reaction

Step	Temperature	Time
Hot Start	98°C	Hold
Initial denaturation	98°C	30 sec
Denaturation	95°C	30 s
Annealing	47°C	60 s
Extension	72°C	6,5 min
DenaturationII	95°C	30 s
AnnealingII	68°C	60 s
ExtensionII	72°C	6.5 min
Final extension	68°C	5 min

• first steps: 15x

• second step: 25x

• program: igbBio2

Result:

• three different PCR samples

• • PCR sample 1: pARW071 + primer 77/78 = mdnABCDE71

• • PCR sample 2: pARW089 + primer 77/79 = mdnABCDE89+T7

• • PCR sample 3: pARW089 + primer 77/80 = mdnABCDE89

Further Tasks:

• gel electrophoresis

• PCR clean up

• restriction enzyme digestion

• ligation

• transformation

Gel extraction and purification of pUP089 (vector) and Lib-2/loop(Insert/PCR)

Investigators: Steffi

Materials:

• Restriction products of pUP089 (vector, 10196 bp) and Lib-2/loop(Insert/PCR) from Niels (2011-08-29)

• Agarose (BioRad)

• 1x TAE

• gel red

• loading dye (promega)

• 1kb DNA ladder (Promega)

• 100bp DNA ladder (Fermentas)

Protocol:

• 1 x 1%: 0.5 g agarose + 50 ml 1xTAE buffer, 2 µl gel red

• 1 x 1.5 %: 0.75 g in 50 ml 1xTAE, 2 µl gel red

Results:

• gel 1 (1.5%)

lane	sample	volume in µl	Expected size in bp
M1	1kb DNA ladder (Fermentas)	10	-
M2	100bp DNA ladder (Fermentas)	10	-
1	Lib-2 (A)	30	161
2	Lib-2 (B)	30	161
3	Loop	30	161

• gel 2 (1%)

lane	sample	volume in µl	Expected size in bp
M1	1kb DNA ladder (Fermentas)	10	-
1	pUP089 (A)	30	10035
2	pUP089 (B)	30	10035

Conclusions:

• cut out bands from Lib-2 (B), Loop, pub089 (A) and pub089 (B)

Gel extraction

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

• Elution with 50µl NE-Buffer
  

NanoDrop: Concentrations

• Lib-2 (B) - digested - purified: 5,8 ng/µl

• Loop - digested - purified: 3,9 ng/µl

• pUP089 (A): 12,9 ng/µl

• pUP089 (B): 23,4 ng/µl
  

Further tasks:

• dephosphorylation of pub089 (B)

• ligation

• transformation

Dephosphorylation and Ligation of pUP089 (vector) and Lib-2/loop(Insert/PCR)

Investigators: Nadine, Steffi

Dephosphorylation of pUP089 (B)

• add 5µl Multicore 10x Buffer (Promega)

• add 1µl Thermosensitive Alkaline Phosphatase (Promega)

• incubate at 37°C for 15 min

• heat inactivation at 74°C for 15 min
  

Ligation

• purified and dephosphorylated pUP089 (B) from 2011-08-30 (vector)

• purified Lib-2 (B) (insert)

• purified Loop (insert)

Insert	[µl]	Vector	[µl]
Lib-2 (B)	1.8	pUP089	10
Loop	2.7	pUP089	10

• 3 µl 10x Buffer

• 1 µl T4 Ligase

• add 30 µl H2O

• 1h, RT
  

Further tasks:

• transformation

Transformation of pUP089+library2

Investigators: Niels

Aim: Transformation of Ligation

Materials:

• competent E. coli cells (XL1-Blue, 2011-08-27, 1xSteffi, 2xSabine)

• ligation products: pUP089+Lib2 (2011-08-30, Steffi)

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:

• 3 plates w/ Kan: mdn-lib2

sequencing of mdnC, mdnD and mdnE BioBrick

Time: 2011-08-30,14:00-14:30

Investigators: Nadine

Materials

Sequencing by GATC

• C2 (mdnC Klon 2, Steffi, 2011-8-29)

• C3 (mdnC Klon 3, Steffi, 2011-8-29)

• D1 (mdnD Klon 1, Steffi, 2011-8-29)

• D3 (mdnD Klon 3, Steffi, 2011-8-29)

• E2 (mdnE Klon 2, Steffi, 2011-8-29)

check plates - resistance of competent E.coli XL1 blue cells

Investigators: Nadine, Steffi

Aim: check resistance of competent cells - E.coli XL1 blue (from 2011-08-27 and 2011-08-29, Steffi)

Materials:

• agar plates from competent cells - E.coli XL1 blue from 2011-08-29 (Kat/ Ste)
  

Results:

• all competent cells - E.coli XL1 blue grow on agar plates with Tetracycline

• no E.coli XL1 clones on agar plates with Ampicillin, Kanamycine, Chloramphenicol

Conclusions:

• competent cells - E.coli XL1 blue work and can be used for transformation

Assembly PCR for TEV and 14_3C

Aim: get the side-directed mutated TEV and 14_3C fragment

Methode:

Primer TEV:

(1) f_TEV_iGEM

(2) r_TEV_iGEM_BamHI

Primer 14_3C:

(1) f_14_3C_iGEM

(2) r_14_3C_iGEM_BamHI

Methode:

PCR

• Template: 1 µL (TEV or 14_3C <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 52°C

Extend: 1 min 72°C

• 25x:

Denat: 1 min 94°C

Anneal: 1 min 69°C

Extend: 1 min 72°C

• Final Extend: 10min 72°C

Further Tasks:

PCR purification to check the correct sizes of fragments

Digestion of created pPDV089 for deletion of kanamycin gene

Investigators: Sabine

Aim:

• inactivation of kanamycin resistence gene

• reason: helper plasmid contains also kanamycin restistence gene

• enable selection of E. coli cells containing both helper plasmid and phage display vector

Reaction components:

• 4 µl/2µg pPDV089 (from clones 3F6, 3F8, 2S7 and 2S14)

• 1 µl restriction enzyme NsiI

• 2 µl buffer 3

• 13 µl water

Reaction conditions:

• 3 h at 37°C

Digestion of PCR products geneIII and mdnA for cloning into pSB1C3

Investigator: Sabine

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

Material/Method:

• 15 µl PCR product mdnA

• 1 µl restriction enzyme XbaI

• 1 µl restriction enzyme PstI

• 2 µl NEB 10x buffer 2

• 0,2 µl BSA

• 0,8 µl water

• 1,5 h, 37°C

• 15 µl PCR product geneIII

• 1 µl restriction enzyme XbaI

• 1 µl restriction enzyme PstI

• 2 µl NEB 10x buffer 2

• 0,8 µl water

• 0,2 µl BSA

• 1,5 h, 37°C

• 15 µl PCR product geneIII

• 1 µl restriction enzyme NgoMIV

• 1 µl restriction enzyme PstI

• 3 µl NEB 10x buffer 1

• 1,5 h, 37°C

• 15 µl PCR product mdnA

• 1 µl restriction enzyme XbaI

• 1 µl restriction enzyme AgeI

• 3 µl NEB 10x buffer 1

• 0,2 µl BSA

• 1,5 h, 37°C

Further Tasks:

• gel electrophoresis for purification

• ligation of mdnA and geneIII into pSB1C3

Digestion of PCR product geneIII and vector pARW089

Investigator: Sabine

Aim: pARW089 containing geneIII but not mdnA

Material/Method:

• 15 µl PCR product geneIII

• 1 µl restriction enzyme SfoI

• 1 µl restriction enzyme AatII

• 2 µl NEB 10x buffer 4

• 1 µl water

• 1,5 h, 37°C

• 6 µl/2µg pARW089

• 1 µl restriction enzyme SfoI

• 1 µl restriction enzyme AatII

• 1 µl NEB 10x buffer 4

• 1 µl water

• 3 h, 37°C

Further Task:

• purification

• ligation

Ligation of geneIII into pARW089

Investigator: Sabine

Aim: cloning of geneIII into pARW089

Material/Method:

• 1,5 µl PCR product geneIII (14,5 µg/µl)

• 15,5 µl pARW089 (11 ng/µl)

• 1 µl T4 ligase buffer (Fermentas)

• 2 µl T4 Ligase (Fermentas)

• 1 h at room temperature

Further task: transformation

Re-ligation of NsiI-digested pPDVs089

Investigator: Sabine

Aim: create pPDV089 without kanamycin resistence

Material/Method:

• 17 µl NsiI-digested pPDVs089 (3F6, 3F8, 2S7 and 2S14, DNA from upper and lower bands)

• 1 µl T4 ligase buffer (Fermentas)

• 2 µl T4 Ligase (Fermentas)

• 1 h at room temperature

Further task: transformation

Ligation of geneIII and mdnA into pSB1C3

Investigator: Sabine

Aim: pSB1C3 containing mdnA, geneIII and mdnA/geneIII

Material/Method:

• 12,5 µl PCR product geneIII (10 µg/µl)

• 4,5 µl pSB1C3 (41 ng/µl)

• 2 µl T4 ligase buffer (Fermentas)

• 1 µl T4 Ligase (Fermentas)

• 1 h at room temperature

• 10,8 µl PCR product mdnA (7 µg/µl)

• 6,2 µl pSB1C3 (41 ng/µl)

• 1 µl T4 ligase buffer (Fermentas)

• 2 µl T4 Ligase (Fermentas)

• 1 h at room temperature

• 4,5 µl PCR product mdnA (7 µg/µl)

• 10 µl PCR product geneIII (7 µg/µl)

• 2,5 µl pSB1C3 (41 ng/µl)

• 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:

• pPDV089 without kanamycin resistence (3F6, 3F8, 2S7 and 2S14)

• pARW089 containing only geneIII (no mdnA)

• 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

gel electrophoresis of TEV and 14_3C

Aim: check sizes

Method:

80 V

Further Tasks:

PCR purification

81th Labday 2011-08-31

Ligation of geneIII into pARW089

Investigator: Sandrina, Laura

Aim: cloning of geneIII into pARW089

Material/Method:

• 2 µl PCR product geneIII (14,5 ng/µl)

• 15 µl pARW089 (16 ng/µl)

• 1 µl T4 ligase buffer (Fermentas)

• 2 µl T4 Ligase (Fermentas)

• 1 h at room temperature

Further task: transformation

Transformation of pARW089 containing only geneIII (no mdnA) in E.coli

Investigator: Sandrina, Laura

Aim:amplification of vectors

Method:

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

• incubation 20 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 kanamycin
  

• storage over night at 37°C
  

Further tasks:
control cell clones

overnight culture of picked E. coli clones transformed with 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:

• 5 clones from pPDV089_2S14 (ampicillin)

• 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
  

over night cultures of pSB1C3+mdnABC/mdnDE

time: 2011-8-31, 18:00

Investigators: Nadine

Aim: check colonies for correct plasmid

Materials:

• agar plates from 2011-8-25, Niels (fridge):

• • pSB1C3+mdnABC

• • pSB1C3+mdnDE

• LB medium

• chloramphenicol (25 mg/ml)

Protocol:

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

• pick colony from plate (from each plate 5 colonies, respectively)

• transfer to LB medium

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

Output:

• over night cultures:

• • pSB1C3+mdnABC

• • • clone 3

• • • clone 4

• • • clone 5

• • • clone 6

• • • clone 7

• • pSB1C3+mdnDE

• • • clone 1

• • • clone 2

• • • clone 3

• • • clone 4

• • • clone 5

Further tasks:

• miniprep

• test digest (for protocol see 2011-8-29)

Assembly PCR for 14_3C, PCR of AraC and TEV

Aim: get the side-directed mutated TEV and 14_3C fragment

Method:

Primer TEV:

(1) f_TEV_iGEM

(2) r_TEV_iGEM_BamHI

Primer 14_3C:

(1) f_14_3C_iGEM

(2) r_14_3C_iGEM_BamHI

Methode:

PCR

• Template: 1 µL (TEV or 14_3C <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 52°C

Extend: 1 min 72°C

• 25x:

Denat: 1 min 94°C

Anneal: 1 min 69°C

Extend: 1 min 72°C

• Final Extend: 10min 72°C

Further Tasks:

PCR purification to check the correct sizes of fragments