Team:Stockholm/30 July 2010

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Nina

IPTG induction on IgG protease-troubleshooting

I have had trouble overexpressing IgG protease (ideS), thus I ran two coomassie gels on IPTG treated BL21 samples. The gels were prepared according to our SDS-PAGE mixtures under protocol.

The IPTG treatment was carried out as once before. As a positive control I treated bFGF in its original vector with IPTG, since I knew it has been overexpressed previously. In addition I had each sample in two versions, such as one with and one without sonication. This was to investigate if the results would have any effect of the sonication. However, I don't recommend not sonicating samples since I observed they get very viscous when loading on to gels.

Arrangement on gels:

Andreas

DNA concentration measurements

Continued from 17/7 samples.

DNA concentrations

Sample	DNA conc (ng/μl)	A₂₆₀/A₂₈₀
pSB1C3.BBa_J18930 A	133.5	1.92
pSB1C3.BBa_J18930 B	37.84	1.95
pSB1C3.BBa_J18931 A	139.9	1.97
pSB1C3.BBa_J18931 B	51.21	2.04
pSB1C3.BBa_J18932 A	22.36	2.29
pSB1C3.BBa_J18932 B	79.83	1.95
pSB1C3.SOD A	120.6	2.02
pSB1C3.SOD B	40.32	1.95
pSB1C3.yCCS A	104.1	1.95
pSB1C3.yCCS B	86.70	1.98

Verification of yCCS site-directed mutagenesis

Site-directed mutagenesis performed by Mimmi, and colony PCR using pSBx VF2 and VR primers. Successful mutagenesis (i.e. removal of the unwanted EcoRI site) tested by digestion of colony PCR amplicons.

Samples (carried on pSB1C3)

yCCS B1	non-mutagenized controls
yCCS B2	non-mutagenized controls
yCCS A	mutagenized clones
yCCS B
yCCS C
yCCS D
yCCS E
Control	Blank

Digestion

17 μl dH₂O
2 μl 10X FastDigest Green buffer
10 μl plasmid DNA
1 μl FastDigest EcoRI

Incubation: 37 °C, 25 min

Gel verification

1 % agarose, 80 V, 50 min (?)

Loaded amounts:

2 μl GeneRuler 1 kb ladder
4 μl undigested sample
6 μl digested sample.

Gel verification of yCCS site-directed mutagenesis.
1st row: λ - uB1 - dB1 - uB2 - dB2 - uA - dA - uB - dB - λ
2nd row: λ - uC - dC - uD - dD - uContr - dContr - uE - dE - λ
u=undigested; d=digested; λ=GeneRuler 1 kb DNA ladder.

Expected bands

Undigested samples: 1076 bp
Digested samples:
- EcoRI removed: ≈120 bp, ≈955 bp
- EcoRI remaining: ≈120 bp, ≈705 bp, ≈250 bp

Results

≈700 bp bands clearly visible in non-mutagenized samples, while these are absent in the mutagenized samples. Also, weak bands are visible at around 250 bp for non-mutagenized samples.

Results indicate successful removal of the intragenous EcoRI site.

LMWP ligation/construction

LMWP primers diluted to 10 pmol/μl in 100 μl samples

Primer ligation

Ligation tubes

N-LMWP	C-LMWP
1 μl LMWP_N_F1	1 μl LMWP_C_F1
1 μl LMWP_CN_F2	1 μl LMWP_CN_F2
1 μl LMWP_CN_R1	1 μl LMWP_CN_R1
1 μl LMWP_N_R2	1 μl LMWP_C_R2
11 μl dH₂O	11 μl dH₂O
4 μl 5X Rapid Ligation buffer	4 μl 5X Rapid Ligation buffer

Incubation

95 °C - 10:00
60 °C - 5:00
37 °C - 5:00
Temp. lowered to 22 °C, samples collected by centrifugation
Added 1 μl Fermentas T4 ligase
22 °C - 15:00
Cooled down on ice.

LMWP insertion

Insert = LMWP ligated from above

Insertion performed in two different vector:insert ratios:

1:10
1:100

N-LMWP & C-LMWP
1:10	1:100
1 μl insert (0.5 pmol)	10 μl insert (5.0 pmol)
2 μl pSB1C3 (0.05 pmol)	2 μl pSB1C3 (0.05 pmol)†
4 μl 5X Rapid Ligation buffer	4 μl 5X Rapid Ligation buffer
12 μl dH₂O	3 μl dH₂O
1 μl T4 ligase	1 μl T4 ligase

†For C-LMWP 1:100, pSB1A3 vector was used instead of pSB1C3.

Incubation in 22 °C, 15 min.

Transformation of ligated vectors into Top10; transformation volume 3 μl. Transformed cells plated and grown on LB agar with appropriate antibiotic 37 °C ON. Ligation samples saved in -20 ° for possible gel verification.

ON cultures

Verified pSB1C3.yCCS A & B clones were inoculated in both 5 ml LB + Cm 25 and 3 ml LB Cm 25. Grown ON in 37 °C/250 rpm and 30 °C, respectively.

Four clones (A, B, C & D) were picked from Mimmi's mutagenized MITF plate and grown ON in 5 ml LB + Amp 100 in 37 °C, 250 rpm to attempt verification of site-directed mutagenesis. Also, two slightly red colonies were discovered on her plate. These were also picked (RED A & B) and grown under same conditions.