Team:Pitt/Skin Probiotic/Cathelicidin/Results

From 2014.igem.org

Results for Cathelicidin

mRFP1 Part:

Lanes: 1 2 3 4 5 6 7	In Lane 1 of this gel is the 1 kb+ ladder. Lanes 3, 5, and 7 all contain the mRFP1 plasmid digested with XbaI and PstI. Each lane has two bands. The first at around ~2100 bp corresponds to the vector backbone. The second is a little above 700 bp, and corresponds to mRFP1 (770 bp).
Blue Promoter (FixK2) Part: Lanes: 1 2 3 4 5	Lane 1 has the 1 kb+ ladder. Lanes 4 and 5 have the FixK2 part digested with XbaI and PstI. The band seen between 200 and 300 bp corresponds to FixK2 (250 bp).
Blue Sensor (YF1 and FixJ) Part: Lanes: 1 2 3	In Lane 1 of this gel is the 1 kb+ ladder. Lanes 3, 5, and 7 all contain the mRFP1 plasmid digested with XbaI and PstI. Each lane has two bands. The first at around ~2100 bp corresponds to the vector backbone. The second is a little above 700 bp, and corresponds to mRFP1 (770 bp).
Hsp60 (no RBS) and Hsp60 (RBS) parts: Lanes: 1 2 3 4 5 6	Lanes 1 and 5 have the 1 kb + ladder. Lanes 2, 3, and 4 have the Hsp60 promoter without RBS (~350 bp). Lanes 6 and 7 have the Hsp60 promoter with RBS (~380 bp).
Blue Promoter (FixK2) – mRFP1 – Cathelicidin part:	Lane 1 contains the 1 kb+ ladder. Lane 2 contains the Blue Promoter – mRFP1 – Cathelicidin part (~1100 bp).
Hsp60 (no RBS) – Blue Sensor (YF1/FixJ) part:	Lane 2 contains the 1 kb+ ladder. Lane 1 contains the Hsp60 (no RBS) – Blue Sensor part (~2200 bp). This part is the larger band, with the vector backbone (~2100 bp) being the smaller.
Blue Promoter (FixK2) – mRFP1 – hsp60 (no RBS) – Blue Sensor (YF1/FixJ) part:	Lane 1 of this gel is the 1 kb+ ladder. Lane 5 contains the Blue Promoter – mRFP1 – hsp60 (no RBS) – Blue Sensor Part. The part is the band at (~3100 bp).

Conclusions

Each of the individual parts was successfully isolated, and all of the part constructs were either completed or partially completed. However, we ran into difficulties during some of our ligations, resulting in delays and failure to fully complete the project. If we had more time, we are confident that we could have solved these problems and that the project would have reached completion. A future goal of the project would be to complete the four parts listed in the introduction, and then to test these parts first in E. coli and then in P. Acnes.

The testing would involve several layers of depth. First would be running each part on an agarose gel and looking for the correct band size. Following that would be testing each part that contains mRFP1 using a fluorescence assay. Additionally, western blots could be performed to look for both the presence of mRFP1 and Cathelicidin in the cells expected to have them.

Testing the parts in P. Acnes would be a little more complicated because it requires ligation of the parts into a separate plasmid that is optimized for growth in P. Acnes. For this reason, we had planned on using the plasmid pBRESP36a, which had shown success. in other Propionibacterium. This plasmid would first need to be modified to remove the BioBrick restriction sites and to contain the prefix and suffix.