Team:Groningen/Labwork/23 July 2013



Run a gel with the PCR products of silk without strep + signal sequence.
The PCR of silk without strep + signal sequence is successful. A new 1.5% agarose gel is made to do gel purification. Did the gel purification. When checked over gel a faint band is seen for some of them. So the samples are combined and concentrated. Concentration is very low. So a PCR is made with the use of this PCR product. Run a gel with the purified PCR product of silk strepF-R and silk F-strepR.
The products are present in 2/3 samples for each silk type. These are combined and concentrated. Concentrations of 18.1 and 16.1 ng/ul are obtained. So a restriction digestion can be made. Made a restriction digestion for the signal sequences. These look fine when checked over gel. Made a new restriction digestion for silk strepF-R and silk F-strepR. Run a gel to determine if the restriction is performed correctly. There are bands present on gel, but only in very low concentrations.


Made a restriction digestion for silk strepF-R and silk F-strepR. No bands are seen on gel.


E. coli with the BBa_818000 backbone that had to grow overnight grew.
Sample Composition BBa_k818000 from growth
1 4 mL LB, 8 uL Amp iGEM 2012 Yes
2 4 mL LB, 8 uL Amp iGEM 2012 Yes
3 4 mL, LB 8 uL Amp (negative control) iGEM 2012 No
4 4 mL LB, 8 uL Amp, 4 uL IPTG iGEM 2012 Yes
5 4 mL LB, 8 uL Amp, 4 uL IPTG iGEM 2012 Yes
6 4 mL, 8uL Amp iGEM 2013 Yes
7 4 mL, 8 uL Amp, 8uL IPTG iGEM 2013 Yes
8 4 mL, 8 uL Amp (negative control) iGEM 2013 No
All suspensions were white. Decided to do a miniprep + digestion to see is plasmid was present. Samples 1 and 6 were chosen, 1 to check if renewed 2012 inoculation was a success and 6 to check if our stock had a the backbone. Upon down spinning the cells (first step of miniprep) a pinkish color was detected in the pellet of cell sample 6. This suggest that the BBa_k818000 backbone was in there after all. Sample 1 showed a less conclusive result. The cells weren't pure white, but also didn't display the same level of pinkness as sample 6.
Fast restriction digestion:
Cut the DNA from the miniprep with EcoRI and PstI restriction enzymes, to check if the gene is there.
If all goes well to bands should be visual on gel on at 5191 bp and on at 1110 bp.
Tubes with DNA and enzymes were placed in the 37C incubator for 1.5 hours. A gel was run at 90V for 35 min.
Both samples showed bands at the theorized height of 1110 bp and 5191 bp. Indicating that the RFP gene and the backbone are present in both cultures.
Did a inoculation of the promoter + backbone plate. Colony 1 was picked and selected for with 8uL Amp in 4 mL LB, cells are to grow overnight in the 37 C shaker. Also a negative control was made.


Plasmid DNA is isolated from the 4 inoculations prepared yesterday.
The isolated plasmid is digested with EcoRI and BamHI to check whether the transformants contain the expected construct.
The gel shows bands at the expected height: 7156bps and 1483bps.

The ligation worked: all the 4 tranformants tested show positive results.

In order to further characterize the new backbone (derived from iGEM Munich 2012 BBa_K823023) the latter is transformed into E. Coli DH5α and the cells are plated on LB + Ampicillin with and without IPTG (1mM). The plates are incubated overnight at 37°C.