Team:Heidelberg/Tyrocidine week22 interms

From 2013.igem.org

Tyrocidine-Indigoidine-fusion extended

As the parts submission had priority last week, we now continued to work on the further constructs for the Tyrocidine-Indigoidine fusion. There were samples for pPW06, pPW09, pPW10 and pPW11. Colonies in DH10β or TOP10 exist for constructs pPW06, pPW09, pPW10, pPW11 and pPW12. Colonies in BAP-I exist for constructs pPW06 and pPW09.

pPW06 (Orn-Val-Ind)

All sequencings for pPW06 were positive, however, the best result was obtained for "pPW06 newC". There already existed a plate of BAP-I cells that were transformed with pPW06 newC. Hence colonies were picked, grown in LB+Cm and induced at OD = ~0.5. After less than two hours, cultures turned blue. When the NRP (expected: Orn-Val-Leu) was purified the next morning, the color of the cultures had already become fainly blue-green. Hence the yield was not high enough to conduct a reliable TLC. Therefore, another liquid culture was inocculated with the same cells in order to obtain higher yield. Using this sample, the following TLC was run which shows the expected different running patterns between Indigoidine and our tagged Dipeptide consisting of Ornithine and Valine.

pPW09 (Pro-Leu-Ind)

Though restriction-digest and screening-PCRs were positive, there was one (always the same) mutation occuring in the primer area at the beginning of the construct. There was a guanine being deleted in all of the cases, which lead to a frameshift. As this occured for multiple clones, we believe that the fragments used in Gibson Assembly already carry the mutation and new fragments should be amplified.

pPW10 (Pro-Leu-Val-Ind)

The exact same thing as for pPW09 accounts for pPW10. Hence the procedure will be the same.

pPW11 (Phe-Orn-Leu-Ind)

There were several minipreps sequenced, however, none of them contained the Tyrocidine part of the construct, but only the C-domain and Indigoidine, hence further colonies were picked, screened with colony PCR, grown overnight, miniprepped and restriction digested with EcoRI.

pPW12 (Phe-Orn-Leu-Val-Ind)

As pPW12 was yet not screened or sequenced, colonies were picked and treated the same as the samples for pPW11.

Colony PCRs for pPW11 and pPW12

Restriction digest with EcoRI leads to the expected linearized product for all samples but pPW12G

The left image shows a screening for the Tyc-part (VF2 and IK17), the right one the screening for the Ind-part (KH05 and VR). Samples that were kept for further analysis are: pPW11D and pPW11F (as they are positive for both modules) and pPW12A, pPW12E and pPW12G as the colonies were small and hence the negative result could be due to missing template. In restriction digest however, only pPW11D, pPW11F, pPW12A and pPW12E showed a band at the expected size. The samples were transformed into BAP-I, grown in liquid culture and induced.