Team:SJTU-BioX-Shanghai/Project/Subproject1/Design

Rare-Codon Switch

Design

We design a Rare-Codon Switch controlling protein biosynthesis.

We can control the translation process by controlling how well the ribosome can get through a tandem of rare codons in the target protein's mRNA.

This process can be achieved by controlling different combinations of Modulators and Reporters

• Modulator: control the amount of charged rare tRNAs that recognize these rare codons. The amount of charged rare tRNA is controlled by two elements:
  • rare tRNA
  • aminoacyl tRNA synthetases (aaRS)

• Reporter: control the number of rare codons in the target protein's mRNA.

Rare tRNA Amount

Design

In this part we have overexpressed rare tRNAArg-AGG in the cell. The rare tRNA can recognize AGG codon on the mRNA.

Over-expression of tRNAArg-AGG: tRNAArg-AGG is over expressed under the control of different promoters: trc promoter (induced by IPTG).

This rare tRNAArg can be charged with Arg by native Arginyl-tRNA Synthetase(ArgRS) in E.coli.

放图

Reporter: we have inserted 6 AGG codons after the start codon ATG in the RFP gene.

(这里要放一张图 6AGG-RFP)

Action

When rare tRNAArg-AGG is not over-expressed, RFP expression is hindered. When tRNAArg-AGG is over-expressed, this tRNA can recognize the AGG codon on the mRNA so a large amount of RFP is produced.

放细胞图

Result

结果图

RFP has been largely produced in cells overexpressing tRNAArg. No RFP can be observed in cells without rare tRNA overexpression.

We have successfully controlled protein expression by controlling rare tRNA amount.

aaRS

Number of Rare Codons

Location of Rare Codons

Modulator

1. tRNA Modulator: inducible rare tRNA and constituitive aminoacyl tRNA synthetase (aaRS)

• Over-expression of tRNA^Arg-AGG: tRNA^Arg-AGG is over expressed under the control of different promoters: trc promoter (induced by IPTG) and sulA promoter (induced by UV).
• This rare tRNA^Arg can be charged with Arg by native Arginyl-tRNA Synthetase(ArgRS) in E.coli.

2. aaRS Modulator: inducible aminoacyl tRNA synthetase (aaRS) and constituitive rare tRNA

• tRNA^Asp-AGG: tRNA^Asp with its anticodon mutated to CCU can base pair with rare codon AGG, which is originally the codon for Arg. This tRNA is under the constitutive aspV promoter.
• TDRS: Aspartyl aminoacyl tRNA synthetase (AspRS) without anticodon recognition domain under the control of T7 promoter and lac operator. This modified AspRS can charge Asp to tRNA^Asp-AGG.

Click here to see Modeling.

Reporter

Reporter controls the number of rare codons in the target protein's mRNA. We chose the rarest codon AGG for Arg in E.coli as our controlling element. A tandem of AGG codons is inserted after the ATG codon of reporter gene.

1. Reporter for Qualitative Analysis: A tandem of 6 AGG codons is inserted after the ATG codon of reporter gene RFP and GFP respectively. The fluorescence emitted reflects how well the ribosome can get through a tandem of rare codons in the target protein's mRNA, thus reflecting how well our system works.

2. Reporter for Quantitative analysis: We use luciferase as our reporter gene for quantitative analysis. The amount of luciferase expressed is reflected by the light emitted when luciferase acts on the appropriate luciferin substrate. The light can be measured by luminometer and the quantity is positively correlated with the amount of luciferase and its activity (Learn more...). We use different combinations of number of AGG codons and strength of promoters to characterize regulation[1].

1) bla promoter-luciferase (weaker promoter)

A tandem of 2, 4, 6 or 8 AGG codons is inserted after the ATG codon of wild type luciferase

2) T7 promoter-luciferase (stronger promoter)

A tandem of 2, 4, 6 or 8 AGG codons is inserted after the ATG codon of wild type luciferase

Click here to see Modeling.

Action: Combinations of Modulators and Reporters

Select a Modulator and a Reporter, then click to see results!

Reference

[1]Ulrich Deuschlel., et al., Promoters of Escherichia coli: a hierarchy of in vivo strength indicates alternate structures The EMBO Journal vol.5 no. 11 pp.2987-2994, 1986