Team:MoWestern Davidson/project wetlab

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Completed tRNAs

We decided to use a weak-medium promoter, pBad, to express our suppressor tRNAs.

PBad-tRNA.png

This promoter choice allows us to induce greater expression with L-arabinose. We found that suppressor tRNAs controlled by pBad is not lethal to E. coli and allows for normal culture growth.

Pbad trna.JPG


  • AGGAC
  • AGGAU
  • CCAAU
  • CCACC
  • CCACU
  • CCAUC (9 bp-anticodon)
  • CCAUC (10 bp-anticodon)
  • CCCUC
  • CGGUC
  • CUACC
  • CUACU
  • CUAGC
  • CUAGU




Pbadrbsreporter.JPG

Completed Reporter With Frameshift Suppressor Leaders (FSL)

  • CUAGC-Chloramphenicol Acetyltransferase
  • CCAUC(9)-Red Fluorescent Protein
  • CCAUC(10)-Red Fluorescent Protein
  • CCCUC- Red Fluorescent Protein
  • CGGUC- Red Fluorescent Protein, tetracycline resistance protein
  • CUACU- Red Fluorescent Protein, tetracycline resistance protein
  • CUAGU- Red Fluorescent Protein, tetracycline resistance protein


Pbadrbsreportertrna.JPG


Choosing A Promoter

One important consideration in our project design was choosing the promoter(s) that would induce a notable phenotype. For controlling our suppressor tRNA expression, we wanted a promoter that would transcribe enough tRNA to cause a high probability of suppression but not so much tRNA that cells would become sick. For controlling our reporters with a leading logical clause, we wanted a promoter that would have strong enough transcription to allow more chances for suppression, and thereby more reporter expression.



Promoter Control of Fluorescence Reporters

In order to have a visible expression of RFP or GFP, we constructed parts with RBS-RFP under the control of 4 different commonly-used promoters:

  • pBad (Potential induction with L-arabinose)
  • pLacI (also called pLac. Potential induction with IPTG)
  • pLacIQ (Potential induction with IPTG)
  • pTet

We found that pLac induced with IPTG caused the greatest expression of RFP. This construct, pLac-RBS-RFP, was chosen as our control construct, representing 100% suppression of the engineered frameshift.


RFPFluorescence.png


RFPpellets.jpg


Promoter Control of Drug Resistance Reporters

Plac+RBS+TetR.JPG
PTet+RBS+TetR.JPG