Team:Stockholm/4 October 2010

From 2010.igem.org

Latest revision as of 21:05, 27 October 2010

Home

Project Idea

Lab Work

Results

Modelling

Team

Follow us on and

2010.igem.org

Andreas

Transfer of pEX.nCPP⋅SOD⋅His to BL21

Gel verification

Colony PCR gel verification of BL21 clones carrying pEX.nLMWP⋅SOD⋅His (1) and pEX.nTra10⋅SOD⋅His (2) plasmids.
4 μl λ; 5 μl sample;
λ = O'GeneRuler 1 kb DNA ladder.

Re-run of 2/10 BL21 samples

1. BL21 pEX.nLMWP⋅SOD⋅His: A & B
2. BL21 pEX.nTra10⋅SOD⋅His: A & B

1 % agarose, 120 V

Expected bands:

1. 744 bp
2. 765 bp

Results

1. Both clones verified
2. Clone A verified; weak band for clone B

ON cultures

• 3 ml LB, 30 °C
  1. A (BL21 pEX.nLMWP⋅SOD⋅His)
  2. A (BL21 pEX.nTra10⋅SOD⋅His)

Transfer of nCPP⋅SOD⋅His.RBS.yCCS operon to pEX

Colony PCR

Picked 2 new colonies of each of the two constructs transformed 30/8:

• 5. pEX.nTra10⋅SOD⋅His.RBS.yCCS 1: A & B
• 6. pEX.nTra10⋅SOD⋅His.RBS.yCCS 2: A & B

Standard colony PCR settings

• Elongation time: 2:00

Gel verification

Colony PCR gel verification of pEX.nCPP⋅SOD⋅His.RBS.yCCS operon clones.
4 μl λ; 5 μl sample;
λ = O'GeneRuler 1 kb DNA ladder.

0.8 % agarose, 100 V

Expected bands:

• 5. 1553 bp
• 6. 1553 bp

Results

• 5. Relevant band for clone B; too large insert (double?) for clone A.
• 6. Relevant band for clone B; too large insert (double?) for clone A.

ON cultures

• 5 ml LB, 37 °C, 250 rpm
  • pEX.nTAT⋅SOD⋅His.RBS.yCCS 2: A
  • pEX.nTAT⋅SOD⋅His.RBS.yCCS 3: B
  • pEX.nTra10⋅SOD⋅His.RBS.yCCS 1: B
  • pEX.nTra10⋅SOD⋅His.RBS.yCCS 2: B
  • pEX.nLMWP⋅SOD⋅His.RBS.yCCS 2: B
  • pEX.nLMWP⋅SOD⋅His.RBS.yCCS 3: B

Verification of pSB1x3 plasmids

Due to some strange growth results with our stock plasmids (pSB1x3.BBa_J04450), I decided to verify their antibiotic resistance. Restreaked clones of the following plasmids (w/ BBa_J04450 inserts) onto Amp 100, Km 50 and Cm 25 plates:

• pSB1A3
• pSB1C3
• pSB1K3
• pSB1AC3
• pSB1AK3

Assembly of His⋅SOD⋅cCPP constructs

Continued from 1/10

Received cCPPs (cTra10, cTAT and cLMWP) in pSB1C3 plasmids, digested with EcoRI and NgoMIV, from Johan.

Ligations

• Vectors:
  1. Dig pSB1C3.cTra10 E+N
  2. Dig pSB1C3.cTAT E+N
  3. Dig pSB1C3.cLMWP E+N
• Insert: Dig pMA.His⋅SOD E+A

• Incubation: 22 °C, 15 min

Transformations

1. pSB1C3.His⋅SOD⋅cTra10
2. pSB1C3.His⋅SOD⋅cTAT
3. pSB1C3.His⋅SOD⋅cLMWP

• Standard transformation
  • 1 μl
  • Cm 25

Nina

Mini prep on IgG_Tra10_N#6

I performed a mini prep on yesterday's inoculated IgG_Tra10_N colony # 6 in 12 ml LB with 24 ul chloramphenicol. The procedure was according to the method described in protocols.

Overday culture of SOD

I put an overday culture of SOD-peX, His-SOD, SOD-His, SOD_TAT_Ntermin and as a positive control yCC-peX from Andrea's glycerol stocks in 12 ml LB with 24 ul amphicillin. I inoculated with a big pipett tip amount in order to have the culture reaching OD 0.6 during the day of laboration.

The reason for why I did this culture was because I had talked to Mimmi about her overexpressions about these constructs and she hadn't got any satisfying results on her gels beside from the yCC sample (which became my positive control). I wanted therefore to check if I would also obtain her type of outcome when overexpressing the proteins of interest.

Gene digestions

I performed digestions of my genes of interest in order to do a gel clean up and follow with a ligation that should yield many positive cloning results.

Digestions:

• Fusion_EA_His # 1 & 3 10 ul
• Fast digestion buffer 10 X 2 ul
• H2O 6 ul
• Restriction enzyme NgoMVI 1 ul
• Restriction enzyme PstI 1 ul (Added after 1.5 h in 37 °C)

• Fusion_NS_His # 2 10 ul
• Fast digestion buffer 10 X 2 ul
• H2O 6 ul
• Restriction enzyme AgeI 1 ul
• Restriction enzyme EcoRI 1 ul (Added after 1.5 h in 37 °C)

• Protein A_EA_His # 5 10 ul
• Fast digestion buffer 10 X 2 ul
• H2O 6 ul
• Restriction enzyme NgoMVI 1 ul
• Restriction enzyme PstI 1 ul (Added after 1.5 h in 37 °C)

• IgG protease_EA_His # 5 10 ul
• Fast digestion buffer 10 X 2 ul
• H2O 6 ul
• Restriction enzyme NgoMVI 1 ul
• Restriction enzyme PstI 1 ul (Added after 1.5 h in 37 °C)

• IgG protease_Tra10_Ntermin # 4 & 6 10 ul
• Fast digestion buffer 10 X 2 ul
• H2O 6 ul
• Restriction enzyme XbaI 1 ul
• Restriction enzyme PstI 1 ul

Incubate in 37 °C for 30 minutes.

Agarose gel on digests

I ran an agarose gel 1 % 100 V on the digested samples in order to check if they have been digested by the enzymes and followed by cutting the bands of interest out of the gel with a scalpel over a UV-lamp for a gel clean up.

Ladder: MassRuler™ DNA Ladder Mix, ready-to-use, 80-10,000 bp

Arrangement on gels:

Gel clean up

I performed a gel clean up of the digested genes. All except IgGEA#5 were digested and cut out of the gel.

I performed the clean up according to the method described in protocols.

The measurements of the cut gel bands, addition of kit solutions and incubation time:

• All bands had a weight of aproximately 0.12 g (120 mg). 120 mg * 3 = 360 ul QXI

• I added 10 ul of QIAEXII to all samples

• All samples were incubated for 5 minutes at RT

• I performed step 11 in the procedure description

Concentration measurments

I measured with a spectrophotometer the concentration of the gel clean up samples.

Sending for sequencing

I sent IgG_Tra10_Ntermin#6 for sequencing. I mixed 15 ul of the miniprep sample and 1.5 ul of Forward bank vector verification primer (VF).

• ASB0045 898

Mimmi

Protein purification

• Using Qiagen Ni-NTA Spin Kit

Buffers

Johan

Miniprep

tra10-bFGF-his, tat-bFGF-his, tat-bFGF-his, lmwp-bFGF-his, his-bFGF-tra10, his-bFGF-lmwp

All ~300 ng/µl

Digest miniprep

2 µl DNA

(1 µl BamHI)

2 µl 10x buffer

15 µl H2O

Gel

tra10-bFGF-his, tat-bFGF-his, tat-bFGF-his, lmwp-bFGF-his, his-bFGF-tra10, his-bFGF-lmwp

Results: All constructs had one correct miniprep

Cut

The parts was cut from its vector to be ligated into pEX vector

5 µl bFGF

2 µl 10x buffer

1 µl XbaI

1 µl PstI

11 µl H2O

Ligation

The vector was first treated with alkaline phosphatase for 60 min.

7 µl bFGF

1 µl pEX

2 µl 10x buffer

1 µl ligase

9 µl H2O

Transformation

3 µl from all constructs was then transformed into top10 cells.

Coomassie gels

I made 2 coomassie gels to be used tomorrow