Team:MoWestern Davidson/project wetlab

Completed tRNAs

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

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.

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

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

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.

Promoter Control of Drug Resistance Reporters