Team:Stockholm/7 October 2010

From 2010.igem.org

Revision as of 17:43, 25 October 2010

Home

Project Idea

Lab Work

Results

Modelling

Team

Follow us on and

2010.igem.org

Andreas

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

Sequencing

15 μl plasmid DNA, 1.5 μl primer

• pEX.nTra10⋅SH.Ry_pEXf: ASB0045 768
• pEX.nTra10⋅SH.Ry_pEXr: ASB0045 769
• pEX.nTAT⋅SH.Ry_pEXf: ASB0045 770
• pEX.nTAT⋅SH.Ry_pEXf: ASB0045 771
• pEX.nLMWP⋅SH.Ry_pEXf: ASB0045 772
• pEX.nLMWP⋅SH.Ry_pEXf: ASB0045 773

• 15 μl plasmid DNA; 1.5 μl primer

Removal of insertion in BioBrick suffixes

An insertion between SpeI and PstI present in IgGp, bFGF, ProtA, yCCS and SOD needs to be removed before submission to the Registry. This will be done by digestion with SpeI (inside insertion) and moving digested gene into a new vector.

Digestions

No sample of SOD available for the moment, so this will be digested after plasmid prep.

• Incubation: 37 °C, 30 min

Gel verification

Gel verification of digested genes for removal of insertion between SpeI and PstI.
3 μl λ; 3 μl sample.
λ = O'GeneRuler 1 kb DNA ladder.

1 % agarose, 130 V

IgGp bFGF ProtA yCCS RFP

Expected bands

• pSB1C3.IgGp (A)
  • Vector: 2050 bp
  • Insert: 990 bp
• pSB1C3.bFGF (B)
  • Vector: 2050 bp
  • Insert: 520 bp
• pSB1C3.ProtA (C)
  • Vector: 2050 bp
  • Insert: 230 bp
• pSB1C3.yCCS (D)
  • Vector: 2050 bp
  • Insert: 800 bp
• pSB1C3.RFP (E)
  • Vector: 2050 bp
  • Insert: 1120 bp

Results
Relevant-sized for all samples showing successful (but incomplete) digestion. Proceeded to gel extraction.

Gel extraction

Loaded remaining 17 μl of each sample on a new 1 % agarose gel. Relevant bands excised by gel extraction and saved in -20 ° for later purification.

ON culture

Set ON culture of pSB1C3.SOD for later plasmid prep.

• 5 ml LB + Cm 25; 37 °C, 225 rpm

Colony PCR of nCPP⋅SOD⋅His.RBS.yCCS operons in BL21

1. BL21 pEX.nTra10⋅SOD⋅His.RBS.yCCS (A & B)
2. BL21 pEX.nTAT⋅SOD⋅His.RBS.yCCS (A & B)
3. BL21 pEX.nLMWP⋅SOD⋅His.RBS.yCCS (A & B)

• Standard colony PCR settings.
• Elongation: 1:30 (too short?)

Gel verification

Colony PCR gel verification of BL21 with pEX carrying the nCPP⋅SOD/yCCS operons.
&3 μl λ; 5 μl sample.
λ = O'GeneRuler 1 kb DNA ladder.

1 % agarose, 130 V

Expected bands

1. 1553 bp
2. 1523 bp
3. 1532 bp

Results
Bands with relevant sizes for all clones.

ON cultures

3 ml LB + Amp 100

1. B
2. B
3. B

Growth curve assay

A growth curve assay will be run to test the toxicity of our CPPs.

ON cultures

5 ml LB + Amp 100; 37 °C, 225 rpm

• BL21, pEX.SOD⋅His
• BL21, pEX.nTra10⋅SOD⋅His
• BL21, pEX.nTAT⋅SOD⋅His
• BL21, pEX.nLMWP⋅SOD⋅His

Nina

Mini prep

I performed a mini prep according to the procedure described in protocols.

The experiment was carried out on colony # 1 from protein A _LMWP_N, _TAT_N, _Tra10_N, Fusion _CPP1, _TAT_C, _CPP3, Fusion_LMWP_N, _TAT_N & _Tra10_N

Protein induction

I prepared a culture of SOD.His (both N & C terminal), SOD_TAT_N, SOD_LMWP_N & SOD_Tra_10 to reach OD600 = 0.6. Then I induced these with IPTG 10 ul/sample.

I took samples at 0, 1, 2 & 3 h, centrifuged 1 min 13000 rpm, resuspended the pellet with 50 ul water and added 50 ul RDSB (with DTT) and stored in a freezer until suitable for loading into an SDS-gel.

Making solution

I made some solutions of RDSB to have in the freezer when loading protein induction on gel.

Werners sample buffer:

• SDS 4.5 g
• Glycerol 15 % 7.5 ml
• Tris pH 7.8 (30 mM) 1.5 ml (1M)
• Bromophenol Blue (a tip of a spoon)
• H2O up to 50 ml

900 ul Werners sample buffer + 100 ul DTT (1M)

Verification of ligation

I ran an agarose gel 1 % 100 V to check if the ligation of IgG_Tra10_N in the pEX vector was successful, but when I looked at the gel there were no bands at all. I must have missed to add the vector and the gene in the ligation.