Week 1

This week we finally started the most important part of our project: the lab work. So of course we had first of all to setup the lab for our needs. Providing schottbottels, flasks, test glasses, ...

This week also the first batch of primers arrived so we started the first PCRs.

The PCR for pnikA was done using the Phusion polymerase, the primers pnikA-E,N,X-for and pnikA-S-rev. The annealing temperature was set for 50°C. The template came from the gDNA of the Escherichia coli strain MG1655. (For more information about the pnikA-system click here). The expected length of the fragment was 280 bp.

The PCR for prcnA was done using the Phusion polymerase, the primers prcnA-E,N,X-for and prcnA-S-rev. Conditions for the Annealing temperatur were at 50°C. The template was again the gDNA of the E.coli strain MG1655. (For more information about the prcnA-system click here). The expected length of the fragment was 230 bp.

The PCR for for the iron-dependant detector are more complex. There are two systems that need to get combined in the same organism to work. (For more information about the iron-dependant-system click here).
For the one system there is a fur-box needed. In cause of inapropriate restriction sites there had to be done two mutagenesis PCRs. Both were done via the Phusion polymerase and with an annealing temperatur of 50°C. The 5'-end PCR was done with the primers Fur_Emut_fwd and Fur_RNS_rev (length of the fragment: 350 bp), the 3'-end PCR with the primers Fur_XR_fwd and Fur_Emut_rev (length of the fragment: 170 bp). The template was from Neisseria meningitidis. Finally we were then abled to do a fusion PCR on the gained fragment with the primers fur_fwd and fur_rev.
Again we used an annealing temperature of 50°C and the Phusion polymerase. The expected length of this PCR was 520 bp. Restriction digest (X/P), into the BioBrick BBa_?????, because this BioBrick is containing a RBS. The backbone was pSB1AK3. This fragment was then was digested again via EcoRI and PstI and ligated with the backbone pSB3C5, as this one is compatible with the backbone pSB1C3, where the other part of the system should be in.
The other part of the system is the promoter norB. The PCR was done with the Phusion polymerase and the primers PnorB_fwd and PnorB_rev. The annealing temperature was 42°C for the 400 bp fragment. The fragment was digested via E/S and fused into several BioBricks containing GFP, lacZ' and luxAB. The fusion with lacZ' seemed to be successful due to a colonyPCR.

The PCRs of pnikA and prcnA were digested with the enzymes EcoRI and SpeI. Together with a reporter they were brought into the backbone pSB1C3 via the Gibson Assembly. The reporters were GFP ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K549001 BBa_K549001]), lacZ' ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K549002 BBa_K549002]] and luxAB ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K549003 BBa_K549003]). The reporters were taken from various BioBricks: GFP from BBa_E0040 ([http://partsregistry.org/Part:BBa_E0040 BBa_E0040]), lacZ' from Ba_J33202 ([http://partsregistry.org/Part:BBa_J33202 BBa_J33202]) and luxAB from the BioBrick BBa_K216008 BBa_K216008]).

Week 2

Still doing PCRs and start restriction digests.

Week 3

First Biobrick is ready!

Week 4

Week 5

Week 6

Week 7

Week 8

After weeks of hard work the last week of our lab work started. As we already prepared our BioBricks for sending them last week there was only the shipment left. So on Monday we said Good-bye and hope that they were going to have a nice journey to Boston. So until Wednesday, the Wiki-freeze, are only a few tasks left: finishing the testing of our BioBricks BBa_K549001 and BBa_K549002 and of course to prepare the last thins for the european jamboree in Amsterdam. Making the presentation, the poster and of course ordering our team-sweaters.

Protocols

Primer