Team:Potsdam Bioware/Labjournal/September part 2

From 2011.igem.org

92th Labday 2011-09-11

miniprep of pSB1C3+mdnABCDE with and without T7 promotor

Time: 2011-09-11

Investigators: Niels, Jessica, Katharina

Materials:

6 overnight cultures

NucleoSpin® Plasmid (NoLid) (Macherey-Nagel)

- Protocol for high-copy plasmids

- elution with 50 µl H₂O

measuring concentration with NanoDrop:

Sample	concentration in ng/µl
mdnABCDE with T7-promotor clone 1	50.6
mdnABCDE with T7-promotor clone 2	10.6
mdnABCDE without T7-promotor clone 1	68.4
mdnABCDE without T7-promotor clone 2	110.2
mdnABCDE without T7-promotor clone 3	108.0
mdnABCDE without T7-promotor clone 4	22.3
mdnABCDE without T7-promotor clone 5(III)	504.0
mdnABCDE without T7-promotor clone 6 (IV)	212.6

miniprep of mdnA foc2 part 2

Time: 2011-09-11, 9:00

Investigators: Jessica, Niels, Katharina

Materials:

1 overnight culture

NucleoSpin® Plasmid (NoLid) (Macherey-Nagel)

- Protocol for high-copy plasmids

- elution with 50 µl H₂O

measuring concentration with NanoDrop:

Sample	concentration in ng/µl
mdnA foc2	155.7

Further Tasks:

give it to the screening group

PCR mdnABCDE with Long PCR Enzyme mix from Fermentas

Time: 2011-09-11

Investigators: Jessica, Katharina

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

Materials

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

DNA polymerases: Long PCR Enzyme Mix (Fermentas)

Template DNA: pARW071 (vector)

dNTPs

water

Primer:

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

Protocol:

mdnABCDE

- 2 µl vector

- 1 µl dNTPs (10 mM)

- 3 µl forward Primer

- 3 µl reverse Primer

- 0.3 µl Polymerase

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

- 35.3 µl water

- total volume: 50 µl

2. PCR program

IGLONG3

first steps: 10x

second steps: 25x

Step	Temperature	Time
Hot Start	94°C	Hold
Initial denaturation	94°C	180 sec
Denaturation	94°C	15 s
Annealing	59	30 s
Elongation	68°C	324 s
DenaturationII	94°C	15 s
AnnealingII	68°C	30 s
ElongationII	68°C	324 s + 2 s per each cycle s
Final Elongation	68°C	600 s

Results:

Further tasks:

PCR purification

digest

ligation

PCR of mdnA with C-terminal myc tag (mycC)

Investigators: Katharina, Jessica

Time: 2011-09-11, 14:00

Material:

pARW089 (~8 ng/µl)

dNTPs

primer 23, 24

HF Phusion Buffer 5x

Phusion Polymerase

Method:

1 µl pARW089

1 µl dNTPs (10 mM each)

2.5 µl forward primer (10µM)

2.5 µl reverse primer (10µM)

10 µl HF Phusion Buffer 5x

0.5 µl Phusion Polymerase

32.5 µl water

total 50 µl

programm IGBIO2

Step	Temperature	Time
Hot Start	98°C	Hold
Initial denaturation	98°C	30 sec
Denaturation	98°C	10 s	10x
Annealing	51°C	20 s
Extension	72°C	20 s
Denaturation	98°C	10 s	20x
Annealing	72°C	20 s
Extension	72°C	20 s
Final extension	72°C	10 min
	4°C	Hold

Result:

PCR product mycN (stored in ?)

Further Tasks:

restriction enzyme digestion

ligation

transformation

Restriction enzyme digestion of mycN and pSB1C3

Investigators: Jessica, Katharina

Time: 2011-09-11

Material:

purified PCR products

- mycN (40.0 ng/µl)

NEB Buffer 4

XbaI

PstI

BSA

- digestion of mycN

- - 20 µl DNA

- - 3 µl Buffer 4

- - 1.5 µl XbaI

- - 2.0 µl PstI

- - 0.3 µl BSA

- - 3.2 µl water

- digestion of pSB1C3

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

- - 3 µl Buffer 4

- - 1.5 µl XbaI

- - 2.0 µl PstI

- - 0.3 µl BSA

- - 11.5 µl water

concentration after digestion

- mycN: 12.2

- pSB1C3: 12.1

PCR Purification of mdnA+mycC, Lib 2 for Phage Display and digested mdnA+mycN

Time: 2011-09-11

Investigators: Niels, Jessica, Katharina

Materials

Macherey-Nagel Nucleo Spin Extract II

- elution in 50 µl

concentration

- mdnA+mycC: 42.6 ng/µl

- Lib 2 for Phage Display: 27.9 ng/µl

- digested mdnA+mycN: 12.2 ng/µl

Gel electrophoresis of pARWIII and pSB1C3

Investigators: Niels, Jessica, Katharina

Time: 2011-09-11

Material:

1% agarose gel

2 µl Gel Red

Fermentas DNA Ladder Mix

lane	Sample	Volume in µl
M	Gene Ruler DNA Ladder Mix (1:10)	12
1	pARWIII	30 + 6 loading dye
2	pSB1C3 (1)	30 + 6 loading dye
3	pSB1C3 (2)	30 + 6 loading dye

fragments were excised and purified using the Macherey-Nagel Nucleo Spin extract II Kit

concentration after purification

- pARWII: 3.9 ng/µl

- pSB1C3 (1): 8.4 ng/µl

- pSB1C3 (2): 12.1 ng/µl

Restriction enzyme digestion of pSB1C3 for control

Investigators: Katharina, Jessica, Niels, Steffi

Time: 2011-09-11

Material:

pSB1C3(2011-06-22) :

concentration: 235,0 ng/µl

digestion of pSB1C3 with Pvu II:

2 µl DNA (235.0 ng/µl (2011-06-22))

1 µl 10xBuffer

1 µl PvuII

6 µl water

- total volume 10 µl

37°C over night

further tast

electrophoretic separation

Ligation of pARWIII + Lib-2 for Phage Display and pSB1C3 + mdnAmycN

Time: 2011-09-11

Investigators: Steffi, Niels, Katharina, Jessica

Material

purified pARWIII (vector) (2011-09-11)

purified Lib-2 for Phage Display (insert) (2011-09-11)

purified pSB1C3 (2011-09-11)

purified mdnA + mycN

Method

ligation of pARWIII + Lib2 for Phage Display

- 1 µl 10x Buffer

- 1 µl T4 Ligase

- 7 µl vector

- 1 µl insert

1h @ RT

ligation of pSB1C3 + mdnAmycC

- 1 µl 10x Buffer

- 1 µl T4 Ligase

- 7 µl vector

- 1 µl insert

1h @ RT

control for each ligation: taking water instead of insert

Further tasks:

transformation

Testing of expression backbones

Time: 2011-09-11

Investigators: Niels, Katharina, Jessica

Material

overnight cultures of pSB1K3_Ara_CFP and pSB1K3_Lac_CFP

Results:

cells didn't grow properly

Further tasks:

repeat with new overnight cultures

Preparing new overnight cultures of expression backbones

Time: 2011-09-11

Investigators: Niels, Katharina, Jessica

Material

glycerol stocks of pSB1K3_Ara_CFP and pSB1K3_Lac_CFP

- stocks weren't prepared correctly, so

plates of pSB1A3_YFP_Ara, pSB1A3_YFP_Lac, pSB1K3_CFP_Ara, pSB1K3_CFP_Lac, pSB1K3_YFP_Lac

Further tasks:

expression test with pSB1K3_Ara_CFP and pSB1K3_Lac_CFP

glycerol stocks of all 5 cultures

Transformation of pARWIII + Lib-2 for Phage Display

Time: 2011-09-11

Investigators:Niels, Jessica, Katharina, Steffi

Aim: Transformation of E. coli cells with ligation of pARWIII + Lib-2 for Phage Display

Materials:

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

ligation products: pARWIII + Lib-2 for Phage Display

Method:

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

incubation 20 min on ice,

heat shock 90 sec at 42°C,

incubation 10 min on ice,

addition of 750 µl LB medium,

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

centrifuge 30 min at 2000 x g

discard the supernatant (till ~100µl media)

plating on LB plate with appropriate antibiotic (Amp)

storage over night at 37°C

Further tasks:

counting colonies

wash colonies from plates

o.n. cultures and miniprep

Colony PCR of E. coli XL1 blue transformed with ligation products of mutated TEV protease, mutated 14_3C protease, AraC form pBAD_iGEM_express, pJC354_ssTorA_XhoI_CS-14_3C_NheI_blaFL and pJC354_ssTorA_XhoI_CS-TEV_NheI_blaFL and analytical AG (1.5%)

Investigators: Stefan, Sascha, Sebastian

Aim: checking for positive clones for further tasks

Methode:

picked clones where incubated over night at 4°C in 1 ml LB media

- 2 µl were used as template for PCR

5 µl Buffer S with 25 mM MgCl2 (purchased by Genaxxon)

2,5 µl each Primer (10 mM, see list below)

1 µl dNTP (each 10 mM)

2 µl Templates

1 µl Taq Polymerase (purchased by Genaxxon)

2 µl 25 mM MgCl2 (purchased by Genaxxon)

34 µl H2O

Total volume: 50 µl

PCR Program:

Initial denat = 5min 98°C

25x

denat: 1min 98°C

anneal: 1min 45sec 55°C

extend: 1min 45sec 72°C

final extend: 10min 72°C

Templates:

TEV1-TEV3 (clones with ligation product of TEV protease, AraC and pJC354_ssTorA_XhoI_CS-TEV_NheI_blaFL, approx. 1,6 kbp)

TEVbb1+TEVbb2 (clones with ligation product of 14_3C protease, AraC and pJC354_ssTorA_XhoI_CS-TEV_NheI_blaFL, approx. 1,6 kbp)

14_3C1-14_3C5 (clones with ligation product of 14_3C protease, AraC and pJC354_ssTorA_XhoI_CS-14_3C_NheI_blaFL, approx. 1,5 kbp)

14_3Cbb1+14_3Cbb2 (clones with ligation product of TEV protease, AraC and pJC354_ssTorA_XhoI_CS-14_3C_NheI_blaFL, approx. 1,5 kbp)

Primer:

Primer for all clones containing TEV protease:

Primer 1 - f_AraC_iGEM_HindIII

Primer 2 - r_TEV_ACCAGC

Primer for all clones containing 14_3C protease:

Primer 1 - f_AraC_iGEM_HindIII

Primer 2 - r_14_3C_tm_XbaI280_A>T

Results:

Clones 14_3C5 (pUP_SG18_ssTorA_CS-14_3C_blaFL_AraC-14_3C5) and Tevbb1 (pUP_SG19_ssTorA_CS-TEV_blaFL_AraC-14_3C1) showed bands with the right size. All other clones showed no visible bands. Precultures are going to be cultivated over night at 37°C 750 rpm in thermo block. PCR will be repeated tomorrow.

100µl of the positive clones are diluted in 5 ml LB media (with chloramphenicol) and incubated over night at 37°C 250 rpm for mini prep of the plasmid DNA.

Picture of GelDoc will follow as soon as possible

Further tasks:

overnight culture for plasmid preparation of pUP_SG18... and pUP_SG19...(sequencing and preparing biobricks)

creating glycerol stock cultures form all positive clones (XL1 blue transformed with pUP_SG18... and XL1 blue transformed with pUP_SG19)

if pUP_SG19... doesnt show any unwanted mutations, this clones needs to be digested with XbaI and PstI and ligated with digested pUP_SG13... (also digested with XbaI and PstI)

survival screening with pUP_SG18... and pUP_SG19... after sequencing without any unwanted mutations

93th Labday 2011-09-12

Preparation of samples for HPLC analysis part II

Time: 2011-09-12

Investigators: Jessica, Katharina

Material:

methanol

PALL Syringe Filter

HPLC vial

Method:

the pellet obtained after speedvac was resuspended in 200µl of 80% methanol

centrifugation for 3 min @ 13000 rpm

samples were filtered using a small PALL Syringe Filter

the filtered sampled was put into a HPLC vial

performance of HPLC

competent cells - E. coli XL1 blue

Investigator: Katharina, Niels, Steffi

Aim: produce competent cells

Materials/Methods:

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

Adjust pH to 5.8 with acetic acid

Filter sterilize the solution

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

Adjust pH to 7.0 with KOH

Filter sterilize the solution

Work always sterile and cold and speedy!

All volumes deal with the common cellline!

Prepare 80 Eppis (1,5?l)

get liquid nitrogen

prepare 5 ml LB-Medium with the specific antibiotic (for 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: 50?l 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, Niels, Steffi

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

Materials/Methods:

- competent XL1 blue-cells from 2011-09-12 (Katharina, Niels, 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

Digest of PCR products mycC, mdnABCDE 71, 89, T7 and pSB1C3

Time: 2011-09-12

Investigators: Jessica, Nicole, Nadja

Materials

PCR products:

- mycC

- mdnABCDE 71

- mdnABCDE 89

- mdnABCDE T7

- pSB1C3

EcoRI, SpeI, BSA, NEB Buffer 4

Reaction mix

for PCR products:

- 10 µl PCR product

- 1.5 µl EcoRI

- 1.5 µl SpeI

- 2 µl Buffer 4

- 0.2 µl BSA

- 4.8 µl water

for pSB1C3:

- 14.8 µl pSB1C3

- 1.5 µl EcoRI

- 1.5 µl SpeI

- 2 µl Buffer 4

- 0.2 µl BSA

Output

digests:

- mycC

- mdnABCDE 71 a

- mdnABCDE 71 b

- mdnABCDE 89 a

- mdnABCDE 89 b

- mdnABCDE T7 a

- mdnABCDE T7 b

- pSB1C3

Further tasks

gel electrophoresis

Purification of mycC, digested mdnABCDE 71-a/b, 89-a/b, T7-a/b and digested pSB1C3 from today

Time: 2011-09-12

Investigators: Jessica, Nicole, Nadja

Materials

Macherey-Nagel Nucleo Spin Extract II

- elution in 35 µl H2O (of mycC and mdnABCDE 71-a/b, 89-a/b, T7-a/b)

- elution in 50 µl NE eluation buffer ( of pSB1C3)

concentration

- mycC: 23,9 ng/µl

- mdnABCDE 71 a: 135,2 ng/µl

- mdnABCDE 71 b: 48,7 ng/µl

- mdnABCDE 89 a: 26,8 ng/µl

- mdnABCDE 89 b: 9,9 ng/µl

- mdnABCDE T7 a: 89,0 ng/µl

- mdnABCDE T7 b: 120,2 ng/µl

- pSB1C3: 32,5 ng/µl

Further tasks

ligation

Ligation of mycC and digested mdnABCDE 71-a/b, 89-a/b, T7-a/b with digested pSB1C3 from today

Time: 2011-09-12

Investigators: Jessica, Nicole, Nadja

Materials

pSB1C3

mycC and digested mdnABCDE 71-a/b, 89-a/b, T7-a/b

Protocol

Components

Component	Molar Ratio	Concentration in ng/ µl	Length in bp	volume
Backbone pSB1C3	1	32,5	2390	fill up to 8µl
Insert: mdnABCDE 71-a	3	135,2	6500	5
Insert: mdnABCDE 71-b	3	48,7	6500	7
Insert: mdnABCDE 89-a	3	28,8	6500	7
Insert: mdnABCDE 89-b	3	9,9	6500	7
Insert: mdnABCDE T7-a	3	89,0	6500	6
Insert: mdnABCDE T7-b	3	120,2	6500	6
Insert: mycC	3	23,9	220	1

Ligation mix

* * for a:

1µl T4 ligase

1µl T4 buffer

fill up with water

- total volume 10 µl

- at 14 C over night

* * for b:

1µl quck ligase

10µl quick ligase buffer

fill up with water

- total volume 20 µl

- at 14 C over night

Further task:

Transformation

ELISA with purified phages

Investigators: Sandrina

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

Method/Materials:

ELISA plate coated with 5 µg/ml anti-c-myc antibody in phosohate buffer (pH 7,4), volume 100 µl/ well

incubate for 4 h at room temperature

blocking in 2 % milk powder in TBS (300 µl/ well)

incubate for 2 h at room temperature

add 100 µl phages, produced in XL1-blu cells and in ER2738 cells diluted 1:5 in TBS-T (0,005%)

incubate shaking for 60 min at room temperature

wash 5 x with TBS-T (0,05%)

add anti-gene-8-antibody (HRP coupled)

incubate shaking for 60 min

wash 5 x with TBS-T

put substrate on the samples

measure in plate photometer

reference: helper phages

Resluts:

x-axis: 1) absorption negative control (helper phages) 2) absorption of phages produced in XL1-blue cells

conclusion: geneIII-myc-mdnA-fusion gene is expressed on the surface of phages produced in XL1-blue cells

at wells incubated with phages produced in ER2738 cells no significant signals were observed

Further tasks:

Phage Display

Send clones 13, 14 (mdnA in pSB) and 7,8 (geneIII in pSB) for sequencing

Investigators: Sandrina

Aim:control ligation of mdnA and geneIII into pSB1C3 for generating biobricks

Method/Materials:

Primer: VR

20 µl with 70 ng/µl DNA

Further tasks:

check alignment

94th Labday 2011-09-13

Preparation of samples for HPLC analysis

Time: 2011-09-13, 8:00

Investigators: Nicole, Jessica, Nadine, Katharina

Aim: Isolation of Microviridin from E. coli

Material:

ON cultures (XL1-blue):

- pSB1C3+mdnABCDE klon 5 (cm resistance)

- pARW089 (kan resistance)

- pARW071 (kan resistance)

LB medium

kanamycin

chloramphenicol

induction solution (content???)

100% methanol

5& methanol

sterile water

Sep-Pak Cartridges

speed vac

Method:

prepare main cultures:

- 150 ml LB + 150 µl appropriate antibiotics

- add 1.5 ml ON culture

- add 150 µl induction solution (time: 8:30)

- incubation at 30°C and 200 rpm to an OD of ???

harvesting and lysis

- pellet was resuspended in 5 ml of sterile water

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

- centrifugation for 10 min @ 11000 rpm

microviridin isolation

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

- cartridges were washed with 2 ml water

- samples were load

- cartridges were washed with 2 ml of 5% methanol

- samples were eluted with 2 ml of 100% methanol

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

Note:

procedure was stopped because we had positive results from first HPLC

Conclusions:

system has not to be induced (only fosmids have to be induced)

HPLC will be repeated w/ remaining sample (2011-9-12), fractions will be collected and analyzed by Mass Spectrometry

HPLC w/ samples from 2011-9-12 (repetition)

Time: 2011-09-13, 8:00

Investigators: Nicole, Nadine, Katharina, Vanessa, Nadja

Aim: this time: collect fraction during the run for MS

Material:

microviridin samples (from pARW089 and pARW071) from 2011-9-12

HPLC (Shimadzu)

HPLC program:

Results:

Conclusions:

Test Expression Backbones: pSB1K3_Ara/Lac_CFP

Time: 2011-09-13

Investigators:
Niels

Aim:

prove of promotors (Lac/Ara)

Materials

pSB1K3_Lac_CFP culture

psB1K3_Ara_CFP culture

Methods

cells from overnight cultre (2011-09-12)were transfer in new media (50ml LB + 50µl Kan (50 mg/ml))

start cell grow in fresh media

- inducing of Ara at 0,6 (OD600)

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

- results negativ

further task

prove of fluorescence by microscope

Expression Backbones: prepare samples for fluorescence microscope

Time: 2011-09-13

Investigators: Nicole,Vanessa, Jessica, Nadine, Nadja

Aim:

plate clones on induced and non-induced plates

also: liquid culture

Materials

pSB1K3_Lac_CFP culture

psB1K3_Ara_CFP culture

pSB1A3_Lac_YFP culture

psB1A3_Ara_YFP culture

LB-Agar

kanamycin

ampicilin

arabinose

IPTG

Digest of pSB1C3 for mdnABCDE

Time: 2011-9-7, 11:00

Investigators: Nadine, Nicole

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

Materials:

BBa_K404304_pSB1C3 (#3), 284.6 ng/µl

EcoRI, SpeI, PstI

Buffer 4

BSA

H₂O

Protocol:

vector (2 batches: batch 1 w/ SpeI and batch 2 w/pstI):

- 5 µl vector

- 1 µl EcoRI

- 1 µl SpeI/PstI

- 2 µl Buffer 4

- 0.2 µl BSA

- 10.8 µl H₂O

- total volume: 20µl

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

Output:

Further tasks:

agarose gel

purification

ligation

Transformation of XL1 with mdnABC and a control with H₂O

Time: 2011-09-14; 23:00

Investigators: Nadine, Nicole, Jessica, Nadja

Materials:

XL1-Blue

ligation product: pSB1C3+mdnABC, Jessica, Nadja, 28.08.2011 resistance: cm

LB-Agar plates

Method:

ligation products:

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

incubation 30 min on ice,

heat shock 60 sec at 42°C,

incubation 5 min on ice,

addition of 750 µl LB medium,

incubation at 37 °C shaking for 60 min,

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

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

Further tasks:

Picking clones for overnight culture

Producing glycerol stocks

Overnight cultures of pARW071_mdnB and pARW089_mdnB

Time: 2011-09-07, 00:00

Investigators: Nadine ,Nicole, Jessica; Nadja

Aim: For characterization via Western blot

Materials

LB medium with Amp

glycerol stocks of of pARW071_mdnB and pARW089_mdnB

Method:

inoculation of 10 ml LB medium with antibiotic

overnight at 37°C

Further tasks:

testing expression

Digestion set

Time: 2011-09-15, 00:00

Investigators: Nadine ,Nicole, Jessica; Nadja

Aim: To build the Biobrick pSB1C3_mdnABCDE

Method:

Digest compositions

sample	mdnE	mdnD	mdnDE	mdnABC
ingrediants	Volume in µl	Volume in µl	Volumein µl	Volume in µl
DNA	5,3	5,0	2,0	1,5
buffer 4	3	3	3	3
BSA	0,3	0,3	0,3	0,3
H2O	18,7	18,7	21,7	22,2
PstI	1,5	1,5	1,5	1,5
XbaI	1,5	-	1,5	-
SpeI	-	1,5	-	1,5

1,5 h at 37°C

Further tasks:

Agarose gel

gel extraction

ligation

Digestion set verification on Agarose Gel, gel extraction and ligation

Time: 2011-09-15, 01:15-05:30

Investigators:Nadine, Nicole, Jessica, Nadja

Materials

digested mdn_ACD

digested mdn_E

digested mdn_D

digested mdn_DE

Production of one 0,8 % gel for mdnABC and mdnDE and one 1% for mdnE and mdnD

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

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

Adding 2 µl gel red to the gel

Loading gels and running

1. 0,8 % gel

lane	Sample	Volume in µl	Expected size in bp
M	Gene Ruler DNA Ladder Mix	15µl (1:100)
1	mdn_ABC	20µl + 4ml 6x loading dye	4819
2	mdn_DE	20µl + 4ml 6x loading dye	2759 (including mdnDE), 2377

2. 1% gel

lane	Sample	Volume in µl	Expected size in bp
M	Gene Ruler DNA Ladder Mix	15µl (1:100)
1	mdn_D	20µl + 4ml 6x loading dye	2934
2	mdn_E	20µl + 4ml 6x loading dye	2394, 2070 (including mdnE)

100V

Ca. 1h

Gel extraction:

using Nucleospin Extract II

- NOTE: accidently elution in collection tube

- second elution with 35 µl NE Buffer

Ligation:

Backbone	Concentration in ng/ µl	Length in bp	volume	Insert	Concentration in ng/ µl	Length in bp	volume
pSB1C3_ABC	4.4	4819	7.3	ED	5.7	2759	9.7
pSB1C3_D	8.2	2934	4.7	E	6.6	2070	12.3

Ligation mix

1 µl T4 ligase

2 µl T4 buffer

insert and vector according to table

total volume 20 µl

at RT for 45 min

Further task:

transformation

Repeated: Digestion set

Time: 2011-09-15, 4:30

Investigators: Nadine ,Nicole, Jessica; Nadja

Aim: To build the Biobrick pSB1C3_mdnABCDE

Method:

Digest compositions

sample	mdnE	mdnD	mdnDE	mdnABC
ingrediants	Volume in µl	Volume in µl	Volumein µl	Volume in µl
DNA	5,3	5,0	2,0	1,5
buffer 4	3	3	3	3
BSA	0,3	0,3	0,3	0,3
H2O	18,7	18,7	21,7	22,2
PstI	1,5	1,5	1,5	1,5
XbaI	1,5	-	1,5	-
SpeI	-	1,5	-	1,5

? h at 37°C

Further tasks:

Agarose gel

gel extraction

ligation

Check sequenced vector pSB1C3 conataining mdnA-myc-geneIII

Investigators: Sabine, Sandrina

Aim:control if biobrick is generated correctly

Method/Materials:

genious

make alignment

Results:

three positive clones (pSB1C3 containing mdnA-myc-geneIII) fusion gene

95th Labday 2011-09-14

Repeated PCR of mdnA for cloning it into pSB1C3

Investigator: Sabine, Sandrina

Time: 2011-09-14,10:30-12:30

Aim:

amplification of mdnA with iGEM-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

Digestion of PCR product mdnA for cloning into pSB1C3

Investigator: Sandrina, Sabine

Aim: cloning of mdnA into pSB1C3

Time: 12:30-14:00

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

Further Tasks:

gel electrophoresis for purification

ligation of mdnA with pSB1C3

Ligation of mdnA with pSB1C3 to generate mdnA as a biobrick

Investigator: Sandrina, Sabine

Time: 2011-09-14, 14:00-15:00

Material/Method:

8,9 µl mdnA (4 ng/µl)

8,1 µl pSB1C3 (17 ng/µl)

2 µl 10x T4 ligase buffer 3

1 µl T4 ligase

1,4 µl water

1 h, room temperature

Further Tasks:

Transformation in XL1-blue cells

Transformation of pSB1C3 containing mdnA in E. coli

Investigator: Sandrina, Sabine

Time: 2011-09-14, 15:00-16:30

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 chloramphenicol

storage over night at 37°C

Further tasks:
control cell clones

Phage Display

Investigator: Sandrina, Sabine

Time: 2011-09-14, 16:00-03:00

Aim:check if unmodified mdnA on the surface of the phages binds to an immobilized protease. interactions of microviridin L with this protease were already detected

Material/Method:

in ELISA plate:

coat wells with protease trypsin in NaCO3 with ph 9,4, for 4 h

blocking in TBS with 3% BSA for 2 h

wash 6x with TBS-T (0,005%)

panning with phages 1:1 helper phages and phages of interest (presenting mdnA-geneIII fusion protein on their surface, 1 h)

wash 6x with TBS-T

elute bound phages with 0,2 M Glycin-HCl, 10 min

neutralize with 1 M Tris (pH 7-8)

mix eluted phages with preparatory culture of XL1-blue cells

incubate 10 min at 37°C without shaking

incubate 50 min at 37°C shaking

plate on amp plates and on kan plates

Further tasks:

check cell growing

Overnight cultures for Western Blot (mdnB)

Investigators:???

Nadine: Who did this?

Time:2011-9-14, ???

Aim:Prepare cultures for Western Blot from pARW089, pARW071

Material:

LB

Amp

glycerol stocks:

- pARW089

- pARW071

96th Labday 2011-09-15

Transformation of pSB1C3_ABC+DE and pSB1C3_D+E into XL1 blue

Investigators:Katharina

Time:7:00-8:45

Aim:Transformation of Ligation

Materials:

competent E. coli cells XL1-Blue

ligation products:

- pSB1C3_ABC+De + ligation control

- pSB1C3_D+E + ligation control

Method:

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

incubation 20 min on ice,

heat shock 45 sec at 42°C,

incubation 3 min on ice,

addition of 750 µl LB medium,

incubation at 37 °C shaking for 60 min,

plating on LB medium with appropriate antibiotic (Cm)

storage @ 37°C

Further tasks:

Picking clones for overnight culture

Producing glycerol stocks

miniprep

sending for sequencing by latest Friday!!

Output:

4 plates with Cm:

- pSB1C3_ABC+DE

- control pSB1C3_ABC

- pSB1C3_D+E

- control pSB1C3_D

Checking fluorescence of expression backbones via fluorescence microscope

Time: 2011-09-15; 7:00-10:00

Investigators:Katharina

Materials

liquid cultures of the expression backbones

- pSB1A3_YFP_Ara

- pSB1A3_YFP_Lac

- pSB1K3_CFP_Ara

- pSB1K3_CFP_Lac

- pSB1K3_YFP_Lac

Method

induction using IPTG and Arabinose, respectively

keep shaking @ 37°C for 2h

check fluorescence of induced and uninduced, control culture with fluorescence microscope (AG Walz)

Results

no fluorescence was detected (just autofluorescence)

Agarose Gel electrophoresis and gel extraction of mdnABC, mdnD, mdnDE, mdnD

Time: 2011-09-15

Investigators:Katharina

Materials

digested mdn_ACD

digested mdn_E

digested mdn_D

digested mdn_DE

Production of one 1% gel

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

Adding 2 µl gel red to the gel

Loading gels and running

1. 0,8 % gel

lane	Sample	Volume in µl	Expected size in bp
M	Gene Ruler DNA Ladder Mix	15µl (1:100)
1	mdn_ABC	30µl + 5µl 6x loading dye
2	mdn_D	30µl + 5µl 6x loading dye
3	mdn_DE	30µl + 5µl 6x loading dye
4	mdn_E	30µl + 5µl 6x loading dye

100V

Ca. 1h then 25V for 2h because of seminar

samples were run out of the gel, but it was decided that we do not need these samples!

Miniprep of several overnight cultures

Investigators: Steffi, Niels, Nadja

Aim:

1. Miniprep:

2 overnight cultures

NucleoSpin® Plasmid (NoLid) (Macherey-Nagel)

- Protocol for high-copy plasmids

- elution with 50 µl elution buffer

measuring concentration with NanoDrop:

Sample	concentration in ng/µl	260/280	260/230
N1	388,8	1,98	2,27
N2	288,3	1,99	2,16
N3	506,9	2,03	2,45
N4	243,0	1,99	2,37
N5	267,2	1,99	2,43
N6	243,4	2,02	2,32
N7	298,9	1,99	2,20
N8	235,4	1,95	2,08
N9	182,2	1,93	2,68
N10	225,5	1,92	2,34
N11	213,8	1,91	2,41
N12	236,9	1,91	2,19
N13	215,1	1,91	2,32
N14	209,2	1,91	2,32
N15	213,0	1,89	2,01
N16	197,6	1,90	2,07

stored in -20°C (red box, expression backbones)

2. Preparation of glycerol stocks:

adding 300 µl glycerol to 700 µl culture

further task

sequencing

sequencing of

Investigators: Niels, Vanessa

Aim:

1. sequencing:

16 clones (miniprep 2011-09-15)

1500 ng in 20 µl:

Sample	DNA [µl]	Water [µl]
N1	3,5	16,5
N2	5	15
N3	3	17
N4	5,5	14,5
N5	5	15
N6	5,5	14,5
N7	4,5	15,5
N8	6	14
N9	8	12
N10	6	14
N11	6,5	13,5
N12	6	14
N13	6,5	13,5
N14	6,5	14
N15	6	14
N16	7	13

seqencing

by GATC

Western Blot of mdnB + pARW071/pARW089

Investigators: Niels,

Aim:

detect mdnB by Immunoblot

Method

overnight cultures spin at max speed

supernatant discard

cells were resuspended in 1 ml 0,5 M Tris-Hcl pH 7,4

cells where broken by Branson Digital Sonifier

- 1s on / 3s off

Nadine: 40 min sonification?!? that is quite long for 1 ml cell suspension...

- 30%amplitude

- for 10 min

spin for 30 min at 4°C by 3220xg

15 µl sample were mixed with 5µl 4x SDS-loading buffer

incubated by 95°C for 5 min

load on gradient gel

- 40V for 30min (max mA)

- 20mA until end (max V)

gel and membrane were equilibrated for 10 minutes in Western blot transfer buffer

blots at 200mA for 1 hour

blocked over night in PBS-T (5% w/v milk powder) at 4°C

Materials

Western blot transfer buffer

15,6 mM Tris

120 mM Glycine

PBS-T (pH 7.4)

140 mM NaCl

2.7 mM KCL

8 mM Na2HPO4

18 mM KH2PO4

0,01% Tween 20

overnight culture of picked E. coli clones transformed with pSB1C3 containing mdnA

Investigators: Sabine, Sandrina

Aim: control ligation of mdnA into pSB1C3

Method/Materials:

10 clones from pSB1C3 with mdnA (chloramphenicol)

5 ml LB medium per clone

storage over night at 37°C and 800 rpm

Further tasks:

test digestion

97th Labday 2011-09-16

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

Investigators: Sascha, Steffi

Aim: check resistance of competent cells - E. coli XL1 blue (from 2011-09-12, Niels, Katharina, Steffi)

Materials:

agar plates from competent cells - E. coli XL1 blue from 2011-09-12 (Kat/Nie/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

File:UP XL1 comp cells.jpg

Conclusions:

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

Antibody detection of mdnB + pARW071/pARW089

Investigators: Steffi

Aim: control if mdnB + pARW071/pARW089 is presented on the membrane

Method:

incubate blocked membrane (Niels, 2011-09-15) for 1 h with primary antibody (anti-mdnB-antibody, 1:10000 in TBS-T)

wash 3x 10 min with TBS-T buffer

incubate for 1 h with secondary antibody (ZAMAK-POD, 1:5000 in TBS-T)

wash 3x 10 min with TBS-T buffer

develop membranes with ECL- Kit

Results:

no signals were seen

Further tasks:

repeat SDS-PAGE for mdnB + pARW071/pARW089

repeat Western Blot and Antibody-Detection

Miniprep of pSB1C3+mdnB

Investigator: Katharina, Steffi

Materials:

liquid culture of pSB1C3+mdnB

NucleoSpin® Plasmid (NoLid) (Macherey-Nagel)

- Protocol for high-copy plasmids

- elution with 50 µl H₂O

measuring concentration with NanoDrop:

Sample	concentration in ng/µl
pSB1C3+mdnB 1
pSB1C3+mdnB 2
pSB1C3+mdnB 3

Further tasks:

sending for sequencing

Miniprep of several overnight cultures pSB1C3+mdnA

Investigator: Katharina, Steffi

Materials:

liquid culture of pSB1C3+mdnA

NucleoSpin® Plasmid (NoLid) (Macherey-Nagel)

- Protocol for high-copy plasmids

- elution with 50 µl H₂O

measuring concentration with NanoDrop:

Sample	concentration in ng/µl
pSB1C3+mdnA 1	323.7
pSB1C3+mdna 2	309.3
pSB1C3+mdna 3	342.8
pSB1C3+mdnA 4	326.7
pSB1C3+mdnA 5	248.8
pSB1C3+mdnA 6	261.8
pSB1C3+mdnA 7	266.6
pSB1C3+mdnA 8	267.7
pSB1C3+mdnA 9	229.7
pSB1C3+mdnA 10	305.1

Further tasks:

test digestion for confirmation

sending for sequencing

Send clones 4,5,8,10 (mdnA in pSB1C3) for sequencing (after test digestion)

Investigators: Sabine, Sandrina

Aim:control ligation of mdnA and geneIII into pSB1C3 for generating biobricks

Method/Materials:

Primer: VF2

20 µl with 70 ng/µl DNA

Further tasks:

check alignment

Repeated Phage Display with another trypsin protease

Investigator: Sandrina, Sabine

Time: 2011-09-16, 9:00-17:00

Aim:check if unmodified mdnA on the surface of the phages binds to an immobilized protease. interactions of microviridin L with this protease were already detected

Material/Method:

in ELISA plate:

coat wells with protease trypsin in NaCO3 with ph 9,4, for 4 h

blocking in TBS with 3% BSA for 2 h

wash 6x with TBS-T (0,005%)

panning with phages 1:1 helper phages and phages of interest (presenting mdnA-geneIII fusion protein on their surface, 1 h)

wash 6x with TBS-T

elute bound phages with 0,2 M Glycin-HCl, 10 min

neutralize with 1 M Tris (pH 7-8)

mix eluted phages with preparatory culture of XL1-blue cells

store over night at 4°C

Further tasks:

check binding of phages by plating them

Digest of pSB1A3 + YFP clone III, ligation with promoters and transformation

Investigators: Nadine, Jessica, Niels

Time: 2011-09-16, 16:00-22:00

Material:

pSB1A3_YFP clone III, 15.08.11, Steffi

digested promoters: Ara 1 (NW, 16.08.11), lac 1(NW, 16.08.11), Ara 2, lac 2

Digest:

30 µl reaction:

- 10 µl pSB1A3_YFP

- 3 µl NEB Buffer 4

- 0.3 µl BSA

- 1.5 µl EcoRI-HF

- 1.5 µl XbaI

- 13.7 µl water

37°C for 1.5 h

Gel electrophoresis:

lane	Sample	Volume in µl
M	GeneRuler™ DNA Ladder Mix (diluted 1:10)	20
1	-	-
2	digest of pSB1A3+YFP	36

[[File:|200px]]

Gel extraction:

using NucleoSpin Extact II

- elution with 50 µl NE buffer

Ligation:

1 µl T4 ligase buffer

1 µl T4 ligase

4 µl backbone

4 µl insert

Transformation:

Nadine: please do not forget to write about the transformation protocol (I think it was Niels´ task?!?)

Output:

amp-LB agar plates

- pSB1A3_YFP_Ara1

- pSB1A3_YFP_Ara2

- pSB1A3_YFP_lac1

- pSB1A3_YFP_lac2

- pSB1A3_YFP control

Further task:

picking clones for overnight plates

98th Labday 2011-09-17

Miniprep of pSB1C3+mdn_ABC+DE and pSB1C3+mdnABC

Investigator: Katharina

Materials:

liquid culture of pSB1C3+mdn_ABC+DE and pSB1C3+mdnABC

Buffer A1, A2, A3 of the Macherey-Nagel Kit

Isopropanol

70% EtOH

Method:

pellet the cells

resuspend in 150 µl Buffer A1

add 150 µl Buffer A2 and invert the tube

incubate @ room temperature for 5 min

add 150 µl Buffer A3 and invert the tube

centrifuge for 5 min @ 11000 rpm

transfer supernatant to a new tube

add 1000 µl isopropanol

vortex and keep at room temperature for 5 min

centrifuge for 5-10 min @ 11000 rpm

discard the supernatant and wash the pellet in 70% ethanol

centrifuge for 5-10 min @ 11000 rpm

discard the supernatant

dry the pellet (either @ room temperature or @ 37°C)

resuspend the pellet in 30 µl sterile water

measuring concentration with NanoDrop:

Sample	concentration in ng/µl	260/280	260/230
mdnABC 1	4013.3	2.07	2.19
mdnABC 2	4549.2	2.06	2.21
mdn_ABC+DE 1	3691.1	2.03	2.20
mdn_ABC+DE	3012.9	1.97	2.07

stored in green mdn-biobrick box (eppis have yellow labels on top)

Checking plates from yesterday (Expression Backones)and picking clones

Investigator: Nadine

Time: 2011-9-17, ~15:00

Materials:

amp-LB agar plates (2011-9-16, Niels)

- pSB1A3_YFP_Ara1

- pSB1A3_YFP_Ara2

- pSB1A3_YFP_lac1

- pSB1A3_YFP_lac2

- pSB1A3_YFP control

LB

ampicillin

Results:

control plate: ~40 clones

ligation plates: ~ 160 clones

Protocol:

5 ml LB

add 5 µl Amp

pick 3 colonies per plate and add to LB medium

Output:

12 overnight cultures:

- pSB1A3_YFP_Ara2, Klon 1, 2 and 3

- pSB1A3_YFP_Ara1, Klon 4, 5 and 6

- pSB1A3_YFP_lac2, Klon 7, 8 and 9

- pSB1A3_YFP_lac2, Klon 10, 11 and 12

Further tasks:

miniprep tomorrow ( DO NOT FORGET THE GLYCEROL STOCKS)

overnight cultures for monday for fluorescence tests

sequencing on monday?!?!

Overnight cultures for Western Blot

Investigator: Nadine

Time: 2011-9-17, ~15:00

Materials:

glycerol stocks:

- pARW089

- pARW071

- pUP089 (#G2)

- pSB1C3+mdnABCDE (#G61 and #G62)

LB

ampicillin

chloramphenicol

kanamycin

Protocol:

10 ml LB

add 10 µl antiobiotics

add cells

incubate overnight at 37°C and 200 rpm

Control of repeated Phage Display with another trypsin protease

Investigator: Sandrina, Sabine

Time: 2011-09-17, 11:00-18:00

Aim:check if unmodified mdnA on the surface of the phages binds to an immobilized protease. interactions of microviridin L with this protease were already detected

Material/Method:

over night culture of XL1-blue cells was infected with eluted phages

1000 cells were infected with 10.000 phages and plated on agar with kanamycin and on agar with ampicillin

control: 1:1 mix of helperphages and produced phages of interest carrying mdnA-geneIII plated also on agar with kanamycin and on agar with ampicillin

Further tasks:

check cell growth

Miniprep of 14_3C2,3,4 and TEv 2,3,4

For better understanding of described experiment see also: [[1]]

Investigators: Sascha, Stefan, Sebastian

Aim:
get plasmids

Methode:

pellet the cells

resuspend in 150 µl Buffer A1

add 150 µl Buffer A2 and invert the tube

incubate @ room temperature for 5 min

add 150 µl Buffer A3 and invert the tube

centrifuge for 5 min @ 11000 rpm

transfer supernatant to a new tube

add 1000 µl isopropanol

vortex and keep at room temperature for 5 min

centrifuge for 5-10 min @ 11000 rpm

discard the supernatant and wash the pellet in 70% ethanol

centrifuge for 5-10 min @ 11000 rpm

discard the supernatant

dry the pellet (either @ room temperature or @ 37°C)

resuspend the pellet in 30 µl sterile water

Further Tasks:

send plasmids for sequenzing

99th Labday 2011-09-18

Miniprep of pSB1A3_YFP Ara und Lac cultures

Time: 2011-09-18

Investigator: Nadja

Materials:

liquid culture: * 12 overnight cultures:

- pSB1A3_YFP_Ara2, Klon 1, 2 and 3

- pSB1A3_YFP_Ara1, Klon 4, 5 and 6

- pSB1A3_YFP_lac2, Klon 7, 8 and 9

- pSB1A3_YFP_lac2, Klon 10, 11 and 12

Buffer A1, A2, A3 of the Macherey-Nagel Kit

Isopropanol

70% EtOH

Method:

pellet the cells

resuspend in 150 µl Buffer A1

add 150 µl Buffer A2 and invert the tube

incubate @ room temperature for 5 min

add 150 µl Buffer A3 and invert the tube

centrifuge for 5 min @ 11000 rpm

transfer supernatant to a new tube

add 1000 µl isopropanol

vortex and keep at room temperature for 5 min

centrifuge for 5-10 min @ 11000 rpm

discard the supernatant and wash the pellet in 500µl 70% ethanol

centrifuge for 5-10 min @ 11000 rpm

discard the supernatant

dry the pellet (either @ room temperature or @ 37°C)

resuspend the pellet in 30 µl sterile water

measuring concentration with NanoDrop:

Sample	concentration in ng/µl	260/280	260/230
pSB1A3_YFP_Ara2 Clone1	718, 8	1,9	1,74
pSB1A3_YFP_Ara2 Clone2	5360, 5	1,9	1,95
pSB1A3_YFP_Ara2 Clone3	5882, 9	2,0	1,86
pSB1A3_YFP_Ara1 Clone4	668	1, 2	1,14
pSB1A3_YFP_Ara1 Clone5	5570, 3	1,9	1,58
pSB1A3_YFP_Ara1 Clone6	7170,3	2,0	1,93
pSB1A3_YFP_Lac2 Clone7	3275,8	5,7	-3.41
pSB1A3_YFP_Lac2 Clone8	4471,6	2,0	1,61
pSB1A3_YFP_Lac2 Clone9	4577,7	2,1	1,97
pSB1A3_YFP_Lac1 Clone10	3535,8	1,9	1,5
pSB1A3_YFP_Lac1 Clone11	3904,6	2,1	2,22
pSB1A3_YFP_Lac1 Clone12	5395,4	2,0	1,91

stored in a new green expression backbones box at -20°C

Further tasks:

overnight cultures for fluorescence measurement

sending for sequencing

Western Blot of mdnB + pARW071/pARW089

Time: 2011-09-18

Investigator: Sebastian, Nadja

Method:

overnight cultures spin at max speed

supernatant discard

cells were resuspended in 1 ml 0,5 M Tris-Hcl pH 7,4

cells where broken by Branson Digital Sonifier

- 2s on / 2s off

- 30%amplitude

- for 2min

spin for 10 min by 17.0 xg

15 µl sample were mixed with 5µl 4x SDS-loading buffer

incubated by 95°C for 5 min

load on gradient gel

- 50V for 30min (max mA)

- 40mA until end (max V)

one gel and membrane were equilibrated for 10 minutes in Western blot transfer buffer

membrane was immobilized with methanol

blots at 200mA for 1 hour

blocked over night in PBS-T (5% w/v milk powder) at 4°C

second gel was stained and shaked over night in Coomassie

Materials:

1. Western blot transfer buffer

15,6 mM Tris

120 mM Glycine

2. PBS-T (pH 7.4)

140 mM NaCl

2.7 mM KCL

8 mM Na2HPO4

18 mM KH2PO4

0,01% Tween 20

Further tasks:

washing steps and treatment with first and second antibody for immunodetection of mdnB

Antibody detection of mdnB + pARW071/pARW089

Investigators: Steffi

Aim: control if mdnB + pARW071/pARW089 is presented on the membrane

Method:

incubate blocked membrane (Nadja, Sebastian, 2011-09-16) for 1 h with primary antibody (anti-mdnB-antibody, 1:10000 in TBS-T)

wash 3x 10 min with TBS-T buffer

incubate for 1 h with secondary antibody (ZAMAK-POD, 1:5000 in TBS-T)

wash 3x 10 min with TBS-T buffer

develop membranes with ECL- Kit

Results:

no signals were seen

Further tasks:

repeat SDS-PAGE for mdnB + pARW071/pARW089

repeat Western Blot and Antibody-Detection

99th Labday 2011-09-20

Miniprep of pSB1A3_YFP Ara-cultures

Investigator: Steffi

Materials:

2 overnight cultures
- pSB1A3_YFP Ara1 I
- pSB1A3_YFP Ara2 II
Buffer A1, A2, A3 of the Macherey-Nagel Kit
Isopropanol
70% EtOH

Method:

pellet the cells
resuspend in 250 µl Buffer A1
add 250 µl Buffer A2 and invert the tube
incubate @ room temperature for 5 min
add 250 µl Buffer A3 and invert the tube immediately
centrifuge for 10 min @ 11000 rpm
transfer supernatant to a new tube
add 0,7 volume of isopropanol
invert the tupe and keep at room temperature for 5 min
centrifuge for 10 min @ 11000 rpm
discard the supernatant and wash the pellet in 500µl 70% ethanol
centrifuge for 10 min @ 11000 rpm
discard the supernatant
dry the pellet (either @ room temperature or @ 37°C)
resuspend the pellet in 30 µl sterile water
measuring concentration with NanoDrop:

Sample	concentration in ng/µl	260/280	260/230
pSB1A3_YFP Ara1 I	3441,1	2,0	2,26
pSB1A3_YFP Ara2 II	4874,4	2,11	2,39

stored in a new green expression backbones box at -20°C

Further tasks:

overnight cultures for fluorescence measurement
sending for sequencing

Analysis of psB1A3_Ava_YFP and psB1A3_IPTG_YFP by fluorescence spectroscopy.

Investigators: Niels, Jessica

Materials:

psB1A3_Ava_YFP over night culture
psB1A3_IPTG_YFP over night culture

Methods:

1 ml culture were transfered in new media
observed growing by measuring OD(600nm)
inducing of promotor by 5 mM Ara or 1 mM IPTG at a OD(600nm) by 0.4-0.5)
measuring of samples every 15 mintes by fluorescence spectroscopy

Results:

further tasks:

analyze by fluorescence microscopy

Analysis of psB1A3_Ava_YFP and psB1A3_IPTG_YFP by fluorescence microscopy.

Investigators: Katharina

Materials:

psB1A3_Ava_YFP over night culture
psB1A3_IPTG_YFP over night culture

Methods:

1 ml culture were transfered in new media
observed growing by measuring OD(600nm)
inducing of promotor by 5 mM Ara or 1 mM IPTG at a OD(600nm) by 0.4-0.5)
measuring of samples by fluorescence microscopy (1h after inducing)

Results:

Sending of BioBricks to MIT

Investigators: Nicole, Nadine, Sebastian

BioBricks

Label number	BioBrick Nickname	Tube label	c [ng/µl]
BBa_K627000	mdnED	1	25
BBa_K627001	mdnA	2	25
BBa_K627002	mdnB	3	25
BBa_K627003	mdnC	4	25
BBa_K627004	mdnD	5	25
BBa_K627005	mdnE	6	25
BBa_K627006	mdnA c-myc gene III	7	25
BBa_K627007	c-myc gene III	8	25
BBa_K627008	AraC TEV protease 1	9	25
BBa_K627009	AraC TEV protease 2	10	25
BBa_K627010	AraC TEV protease 3	11	25
BBa_K627011	AraC 14_3C protease	12	25
BBa_K627012	ssTorA CS-TEV BlaFL	13	25
BBa_K627013	ssTorA CS-14_3C BlaFL	14	25
BBa_K627014	A3_Ara_YFP clone 1	15	25
BBa_K627015	A3_lac_YFP clone 2	16	25

Team:Potsdam Bioware/Labjournal/September part 2

From 2011.igem.org

Contents

92th Labday 2011-09-11

miniprep of pSB1C3+mdnABCDE with and without T7 promotor

miniprep of mdnA foc2 part 2

PCR mdnABCDE with Long PCR Enzyme mix from Fermentas

PCR of mdnA with C-terminal myc tag (mycC)

Restriction enzyme digestion of mycN and pSB1C3

PCR Purification of mdnA+mycC, Lib 2 for Phage Display and digested mdnA+mycN

Gel electrophoresis of pARWIII and pSB1C3

Restriction enzyme digestion of pSB1C3 for control

Ligation of pARWIII + Lib-2 for Phage Display and pSB1C3 + mdnAmycN

Testing of expression backbones

Preparing new overnight cultures of expression backbones

Transformation of pARWIII + Lib-2 for Phage Display

Colony PCR of E. coli XL1 blue transformed with ligation products of mutated TEV protease, mutated 14_3C protease, AraC form pBAD_iGEM_express, pJC354_ssTorA_XhoI_CS-14_3C_NheI_blaFL and pJC354_ssTorA_XhoI_CS-TEV_NheI_blaFL and analytical AG (1.5%)

93th Labday 2011-09-12

Preparation of samples for HPLC analysis part II

competent cells - E. coli XL1 blue

check resistance of competent cells - E. coli XL1 blue

Digest of PCR products mycC, mdnABCDE 71, 89, T7 and pSB1C3

Purification of mycC, digested mdnABCDE 71-a/b, 89-a/b, T7-a/b and digested pSB1C3 from today

Ligation of mycC and digested mdnABCDE 71-a/b, 89-a/b, T7-a/b with digested pSB1C3 from today

ELISA with purified phages

Send clones 13, 14 (mdnA in pSB) and 7,8 (geneIII in pSB) for sequencing

94th Labday 2011-09-13

Preparation of samples for HPLC analysis

HPLC w/ samples from 2011-9-12 (repetition)

Test Expression Backbones: pSB1K3_Ara/Lac_CFP

Expression Backbones: prepare samples for fluorescence microscope

Digest of pSB1C3 for mdnABCDE

Transformation of XL1 with mdnABC and a control with H2O

Overnight cultures of pARW071_mdnB and pARW089_mdnB

Digestion set

Digestion set verification on Agarose Gel, gel extraction and ligation

Repeated: Digestion set

Check sequenced vector pSB1C3 conataining mdnA-myc-geneIII

95th Labday 2011-09-14

Repeated PCR of mdnA for cloning it into pSB1C3

Digestion of PCR product mdnA for cloning into pSB1C3

Ligation of mdnA with pSB1C3 to generate mdnA as a biobrick

Transformation of pSB1C3 containing mdnA in E. coli

Phage Display

Overnight cultures for Western Blot (mdnB)

96th Labday 2011-09-15

Transformation of pSB1C3_ABC+DE and pSB1C3_D+E into XL1 blue

Checking fluorescence of expression backbones via fluorescence microscope

Agarose Gel electrophoresis and gel extraction of mdnABC, mdnD, mdnDE, mdnD

Miniprep of several overnight cultures

sequencing of

Western Blot of mdnB + pARW071/pARW089

overnight culture of picked E. coli clones transformed with pSB1C3 containing mdnA

97th Labday 2011-09-16

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

Antibody detection of mdnB + pARW071/pARW089

Miniprep of pSB1C3+mdnB

Miniprep of several overnight cultures pSB1C3+mdnA

Send clones 4,5,8,10 (mdnA in pSB1C3) for sequencing (after test digestion)

Repeated Phage Display with another trypsin protease

Digest of pSB1A3 + YFP clone III, ligation with promoters and transformation

98th Labday 2011-09-17

Miniprep of pSB1C3+mdn_ABC+DE and pSB1C3+mdnABC

Checking plates from yesterday (Expression Backones)and picking clones

Overnight cultures for Western Blot

Control of repeated Phage Display with another trypsin protease

Miniprep of 14_3C2,3,4 and TEv 2,3,4

99th Labday 2011-09-18

Miniprep of pSB1A3_YFP Ara und Lac cultures

Western Blot of mdnB + pARW071/pARW089

Antibody detection of mdnB + pARW071/pARW089

99th Labday 2011-09-20

Miniprep of pSB1A3_YFP Ara-cultures

Analysis of psB1A3_Ava_YFP and psB1A3_IPTG_YFP by fluorescence spectroscopy.

Analysis of psB1A3_Ava_YFP and psB1A3_IPTG_YFP by fluorescence microscopy.

Sending of BioBricks to MIT

Transformation of XL1 with mdnABC and a control with H₂O