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 a nnealing 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 fur-norB-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 gDNA from Neisseria meningitidis.

Restriction digest (X/P), into the BioBrick [http://partsregistry.org/Part:BBa_J04500 BBa_J04500], because this BioBrick is containing the constitutive promoter lacI and a RBS. The backbone was pSB1AK3. The gained BioBrick is [http://partsregistry.org/wiki/index.php?title=Part:BBa_K549004 BBa_K549004].

The fragment was digested via E/S and fused into several BioBricks containing GFP, lacZ' and luxAB. (PnorB)

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]).

Of course we also had to proliferate our needed BioBricks that we transformend into super-competent cells. From the overnight cultures we did plasmid preps.

Week 2

fur: 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.

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. template was the gDNA of Neisseria meningitidis. The annealing temperature was 50°C for the 400 bp fragment. Unfortunately the PCR gave not the wanted fragment.

PCR for the arsR system: [http://partsregistry.org/Part:BBa_J33201 BBa_J33201] with the Phusion polymerase. The wanted fragment contains a promoter, the ABS part, a RBS and arsR. The expected length is 500 bp. Used primers are parsRENX_fwd and arsRABSS_rev. The annealing temeperature is 50°C. The PCR did not work out. In the next step we tried a gradient PCR to find the ideal annealing conditions for our fragment. The gradient reached from 42-65°C. This time we also changed the polymerase and used Taq. Also this attempt gave not the wanted result. Also the PCR with the Pfu polymerase and an annealing temperature of 50°C gave not the fragment of interest.

PCR for the BioBrick [http://partsregistry.org/wiki/index.php?title=Part:BBa_K549009 BBa_K549009] containing luxCD was made using the Phusion polymerase, the primers luxCENX_fwd and luxDS_rev. The backbone was the plasmid pBBR1-MSC-lux. The annealing temperature for the 2,4 kb fragment was set at 50°C. This PCR did not work out under these conditions. With changing the annealing temperature to 55°C the PCR finally worked as expected.

We also did the PCR for [http://partsregistry.org/wiki/index.php?title=Part:BBa_K549010 BBa_K549010]. This BioBrick is the second part for the BioBrick [http://partsregistry.org/wiki/index.php?title=Part:BBa_K549011 BBa_K549011] together with BBa_K549009. The conditions for the PCR with the Phusion polymerase were 50°C for the annealing temperature, the primers luxEENX_fwd and luxESNP_rev and the backbone pBBR1-MSC-lux. The expected length of the fragment is 1,1 kb. The PCR did not work. For the next PCR we reduced the annealing temperature to 42°C which worked out as wanted.

Week 3

For PnorB we tried a gradient PCR from 40-65°C. The annealing temperatur that was best fitting was 42°C. The PCR was done using the Phusion polymerase. Temolate was the gDNA of N. meningitidis, primers PnorB_fwd and PnorB_rev.

First Biobrick is ready!

digestion of luxCD (ES), luxE (SP) and lacZ' (XP).

For the BBa_K549011 we did a colony PCR for checking if the construct was correct. Conditions were an annealing temperature of 50°C, the Phusion polymerase and the primers were the ones for the BioBricks (BB_fwd and BB-rev).

Week 4

Redoing the PCR for PrcnA under the same conditions as in the first week.

As the PCR for the BioBrick BBa_K549012 never worked, we tried another BioBrick (BBa_J33206) also containing arsR. This time the arsR was provided by B. subtilis instead of E. coli. We did a gradient PCR reaching form 42°C to 67°C using the Phusion polymerase. Another attempt was a gradient PCR from 42-58°C using the same polymerase. The PCR that worked finally out was using not the BioBricks as templates but the gDNA of B. subtilis, but the same PCR conditions.

Digest of BBa_J04500 (SP), GFP (XP), lacZ' (XP) and luxAB (XP).

Week 5

Our first WORKSHOP took place.

On saturday we made video for our iGEM-song: [http://www.youtube.com/watch?v=vVY3pU11ViI check this out]

The second batch of primers arrived in the beginning of the week, so we could finally start with the protein-based metal detectors.

This insert of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K549004 BBa_K549004] was then digested again via EcoRI and PstI and ligated with the backbone pSB3C5 [http://partsregistry.org/wiki/index.php?title=Part:BBa_K549005 BBa_K549005], as this one is compatible with the backbone pSB1C3, where the other part of the system should be in. (PnorB)

rcnA (EcoRI and SpeI digest), GFP from BBa_E0040 (XbaI and PstI digest) and pSB1C3 (EcoRI and PstI digest) were ligated.

Ligation of luxCDE into pSB1C3 ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K549011 BBa_K549011]).

Ligation of the insert of BioBrick [BBa_K549012] into the backbones with GFP, lacZ' and luxAB. Before they were digested via EcoRI and SpeI, the backbone via XbaI and PstI.

Ligation of norB into the backbones of GFP, lacZ' and luxAB. Digest with ES and backbone XP.

Digest of petE2 and pabA via XbaI and AgeI. ligation into pMA-BBFR (digest with XbaI and AgeI).

digest of BBa_K549011 via XP and Jo4500 via SP. ligation and transformation in dh5alpha.

PCR of the fragment petE2 from the cDNA of Arabidopsis thaliana for [http://partsregistry.org/wiki/index.php?title=Part:BBa_K549024 BBa_K549024]. Used were the primers petE2_fwd and petE2_rev, the Phusion polymerase and an annealing temperature of 52°C. The length of the fragment is 530 bp. In the following the fragment was cut via AgeI and XbaI and ligated into pMA-BBFR that was also cut by the same restriction enzymes.

The PCR for [http://partsregistry.org/wiki/index.php?title=Part:BBa_K549020 BBa_K549020] was done with the template BBa_S04034 that contains pabA, the glutamine aminotransferase. Primers were pabA_fwd and pabA_rev using the Phusion polymerase and an annealing temperature of 57°C for the 600 bp long fragment. Following the fragment was digested via AgeI and XbaI, exactly as pMA-BBFR, and both were ligated.

Week 6

We had to redo the PCR for PnorB as there were some tryings to get the part into pSB1C3. But finally we seemed to have succeded. The fusion of PnorB with lacZ' into the backbone pSB1C3 ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K549007 BBa_K549007]) seemed to be successful due to a colonyPCR.

Redoing PCR for PrcnA under the same conditions as in week 1.

Digest of backbones for GFP, luxAB and lacZ' via XP. pMA-BBFR via XA, norB and PrcnA via ES, petE2 and pabA via XA. ligations: petE2 and pabA into pMA-BBFR, norB and PrcnA in the three different backbones containing a reporter. Following transformation in DH5alpha.

This week we also had our interview with biotechnologie.tv about our project. To watch the interview click [http://www.youtube.com/watch?v=-wXGm_Q6Xz8 here].

Digest of the backbones containing GFP, lacZ' and luxAB via EX, BBa_K157000 via AX. J04500 SP, pSB1K3 EP, pSB1C3 EP, pSB3C5 EP.

Week 7

digest of BB 11 via XP and J04500 via SP. ligation and following transformation.

digest BB4 via EP and ligation into pSB3C5 (digest via EP).

digest of the reporter backbones via EX. pMA-BBFR via XA, the insert of BBa_K549004 (fur + lacI + RBS) via EP and nikA via ES. ligations: nikA in the backbones of the different reporters, as well as the insert of BBa_K549004. The STREP-tag, 10xHis-tag, pabA and petE2 into pMA-BBFR.

The STREP and 10XHis-tag were done by heating the two primers (STREP_fwd and STREP_rev, His_fwd and His_rev) at a temperature of 95°C and cooling them slowly down.

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 things for the european jamboree in Amsterdam: Making the presentation, the poster and of course ordering our team-sweaters.

Protocols

All protocols were if not described different were followed according to the protocols on [http://openwetware.org/wiki/Main_Page openwetware].

Primer