IGEM:UNAM-Genomics Mexico/2009/Notebook/Wiki Creation/2011/06/14

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Objectives

  • Design a method for converting proyect text into HTML suitable text.
  • Design in silico the four intergenic operon parts for Gibson Assembly with the Silver_lab ORF's, to be synthesised.

Week meeting

During this meeting we agreed that only Abiel and I will edit the page unless an exceptional situation with immediate editing need is encountered. Since we had already agreed in an notebook like displaying of our page, I agreed in presenting a notebook layout for our wiki.

Our team still has no logo. We agreed in presenting logos for the last time on friday 17. I am presenting one alternative logo.

As part of our experimental procedures we optimized the ORF's of our system according to Rhizobium etli 's Codon usage Adaptation Index CAI. We asked Pamela Silver's Lab for the un-optimized ORF's used in our system to reassemble it without the optimization as a control experiment. However, in order to assemble them in our designed operons through Gibson Assembly we need some complementarity at the end of our sequences. We will send for synthesis the intergenic spacers and the promoters of our system with 3bp ORF overlapping regions (non-optimized). I will work on this as a high priority task, along with Helena and Daniel.

Achievements

  • Intergenic parts design is done.
  • The method design was NOT achieved.

Notes

We discovered a mistake in the sequences we sent for synthesis in the first place. The first gene in the second operon, hydEF, has a Ribosomal Binding Site 27 nucleotides before the start codon AUG. This separation will disrupt completely hydEF translation. We will solve this problem using the pLacZ unoptimized synthesis part wich contains the appropiate spacer. We must design one of the two following primers to generate an overlapping region between the promoter and the optimized hydEF ORF for Gibbson Assembly:

  • A primer designed for the promoter polymerization through PCR with a complementary region with the optimized TAT tag.
  • A primer designed for the optimized synthesis PCR capturing hydEF from it's start codon and having complementarity with the 6bp correct spacer.

I believe the first primer would be the best since its PCR would ampliffy a much smaller part (the promoter) than the later (which has at least to ampliffy all the hydEF ORF for a furhter Gibson Assembly with hydG, or at worst has to ampliffy all the operon to avoid further assemblies).



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