Regulatory

Part:BBa_K2703002:Experience

Designed by: Aurelie Bouin, Victor Sayous   Group: iGEM18_Sorbonne_U_Paris   (2018-10-01)
Revision as of 14:59, 9 October 2018 by Saniya kari (Talk | contribs)

__NOTOC__ This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out. ===Applications of BBa_K2703002=== Our part is compatible with the PhytoBrick MoClo standard. This PhytoBrick was intented to be use as constitutive promoter for the transcription unit of paromomycine in the CARGO of our synthetic retrotransposons for directed evolution on the Chlamydomoas. It is part of our reporter system to characterize the activity and transposition rate.

We couldn’t directly use our Phytobrick for the characterisation because we didn’t have time to construct all the other that are needed for the final assembly. Instead we use a slightly different Phytobrick with the same P PSAD. sequence but with one different fusion site. This part was assembled in a functional Transcription Unit with the resistance gene paromomycine that was as a reporter gene. After transformation in C.reinhardtii D66 the functionality of the promoter was tested by counting the number of chlamydomonas colony resistant to paromomycine (15 ug/ml). Different constructions were made to characterize and compare the functionality of P pSAD . We tested different combination of promoters and 3’UTR.

Name F1 Prom F2 5'UTR F3 Resistance F4 3'UTR F5 pCM-1 GGAG PAR TACT 5’UTRAR AATG Paromycin GCTT TRBCS2 CGCT pCM-2 GGAG PAR TACT 5’UTRAR AATG Paromycin GCTT TPSAD CGCT pCM-3 GGAG PPSAD TACT 5’UTRPSAD AATG Paromycin GCTT TRBCS2 CGCT pCM-4 GGAG PPSAD TACT 5’UTRPSAD AATG Paromycin GCTT TPSAD CGCT

Figure 2: The 4 different devices made to test the functionality of the promoter PPSAD. The construction were made with the MoClo kit made for C.reinhardtii by P.Crozet et al 2018 1.

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