Team:Stockholm/5 October 2010

From 2010.igem.org

Andreas

Transfer of pEX.nCPP⋅SOD⋅His to BL21

Glycerol stocks

BL21 pEX.nLMWP⋅SOD⋅His
BL21 pEX.nTra10⋅SOD⋅His

Assembly of His⋅SOD⋅cCPP constructs

Colony PCR

pSB1C3.His⋅SOD⋅cTra10 A-D
pSB1C3.His⋅SOD⋅cTAT A-D
pSB1C3.His⋅SOD⋅cLMWP A-D

Standard colony PCR settings

Elongation time: 1:30

Gel verification

Colony PCR gel verification of His⋅SOD⋅cCPP constructs in pSB1C3.
4 μl λ; 5 μl sample.
λ = O'GeneRuler 1 kb DNA ladder.

1 % agarose, 120 V

Expected bands

884 bp
854 bp
863 bp

Results
Varying band sized, with the following clones seeming correct:

A
D
B & C

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

Plasmid preps

From 4/10 ON cultures

As I'm still waiting for sequencing results of the operons in pSB1K3, I spun down cells (4,400 x g, 10 min), and saved pellets in -20 °C ON.

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

Nina

Ligation of proteins into peX and CPP containing vectors

Ligations:

Fusion protein-His into three vectors each containing all N-terminal CPPs LMWP, TAT and Tra10.

* Gene: 10 ul 
* Vector: LMWP 0.5 ul (25ng), TAT 1 ul (20 ng) and Tra10 1 ul (20 ng)
* Quick Ligation buffer 2X: LMWP 11.5 ul, TAT 12 ul and Tra10 12 ul
* Quick Ligase: 1 ul

His-Fusion protein into three vectors each containing all C-terminal CPPs LMWP, TAT and Tra10.

* Gene: 6 ul 
* Vector: CPP1 0.5 ul (25ng), TAT 1 ul (20 ng) and CPP3 1 ul (20 ng)
* Quick Ligation buffer 2X: CPP1 7.5 ul, TAT 8 ul and CPP3 8 ul
* Quick Ligase: 1 ul

Protein A-His into three vectors each containing all N-terminal CPPs LMWP, TAT and Tra10.

* Gene: 12.5 ul 
* Vector: LMWP 0.5 ul (25ng), TAT 1 ul (20 ng) and Tra10 1 ul (20 ng)
* Quick Ligation buffer 2X: LMWP 14 ul, TAT 14.5 ul and Tra10 14.5 ul
* Quick Ligase: 1 ul

IgG protease_Tra10_Ntermin_#6 into peX vector

* Gene: 7 ul 
* Vector: peX 1 (25ng)
* Quick Ligation buffer 2X: 9 ul
* Quick Ligase: 1 ul

All ligation mixtures were incubated in 22 °C (in a water bath) for 15 minutes. During these minutes I also thawed competent Top 10 cells (100 ul) on ice for transformation of the ligation samples.

Transformation of ligation products

The procedure was according to the method decribed in protocols. However I thawed Top 10 cells (100 ul) in 15 minutes instead of 10. I added 3 ul of ligation samples into each 100 of Top 10 cells.

Mimmi

Protein purification

Resuspend pellet in

mix
626.85µl	NPI-10/20
3.15µl	imidazole 2M

Add 70µl lysozyme
- Add 14µl DNase 20µg/ml -> 14µg
- Add 0.7µl PMSF 1mM
- Inbcubate on ice for 15-30min

Centrifuge lysate at 12,000xg for 20min at 4°C
- Collect supernatant
- Save 20µl for SDS-Page

PhastGel

well	sample
1	ladder
2	SOD 3h
3	SOD.his 0h
4	SOD.his sup.
5	his.SOD 0h
6	his.SOD sup.

Johan

What went wrong

I got NO colonies on all plates, something is wrong.

Cut

vector with insert, to see if its ligated
vector with insert + BamHI
vector with insret - BamHI
2 µl pEX vector

While running, I talked to Rickard, the guy that yesterday told me that it works to use invitrogen ligase enzyme with fermentas buffer. Further research showed that invitrogen buffer has Pog8000 which fermentas buffer dont have, which probably means that invitrogen ligase needs Pog8000 - which it didnt have in the fermentas buffer

Ligation & transformation

Did another ligation into pEX with fermentas ligase AND buffer, I then transformed 3 µl of all ligations into top10 cells.

Coomassie

Did a coomassie of some constructs from Andreas & Mimmi. SOD, tat-SOD-his, SOD-his, his-SOD and yCCS. 0, 1, 2 and 3h after IPTG induction for all samples.