Part:BBa_K567001

lacI-Ptrc-tRNA(Arg)

The tRNA^Arg is under the control of promoter trc. tRNA^Arg expression is induced by 0.5mM IPTG when the OD600 of the culture reaches 0.3.

This part is used to control the translation process by over expressing tRNA^Arg, and to cooperate with related parts to analyse and characterize three factors in rare-codon switch system.

Control Rare tRNA amount

Design

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

tRNA^Arg-AGG(BBa_K567001): tRNA^Arg-AGG is over expressed under the control of trc promoter (induced by IPTG).

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

RFP-6AGG(BBa_K567017): we have inserted 6 AGG codons after the start codon ATG in the RFP gene.

Action

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

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Result

Fig.1 Confocal Microscope examining RFP expression. RFP has been largely produced in cells overexpressing tRNA^Arg-AGG.

Fig.2 Cells over-expressing tRNA^Arg-AGG emit bright red fluorescence as wild type RFP (first one from the left). Control (first one from the right) exhibits no red fluorescence.

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

Three Factor in Rare-Codon Switch

Number of Rare Codons

In this part we want to explore the influence of the number of rare codons inserted in the mRNA. We have inserted 2, 4, 6, 8 AGG codons respectively after the start codon in luciferase gene. T7 promoter or bla promoter^[1] are used to control target protein mRNA amount. 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

Parts:

• Pbla-Luc-2AGG(BBa_K567004)

• Pbla-Luc-4AGG(BBa_K567005)

• Pbla-Luc-6AGG(BBa_K567006)

• Pbla-Luc-8AGG(BBa_K567007)

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

Parts:

• PT7-Luc-2AGG(BBa_K567008)

• PT7-Luc-4AGG(BBa_K567009)

• PT7-Luc-6AGG(BBa_K567019)

• PT7-Luc-8AGG(BBa_K567010)

Influence of inserted AGG codon number

The influence of different number of rare codons in regulating protein biosynthesis is shown below:

Fig.3 (A) Comparing the influence of different number of rare codon insertions in luciferase production. Pbla-Luc-4AGG (BBa_K567005), Pbla-Luc-6AGG (BBa_K567006) and Pbla-Luc-8AGG (BBa_K567007). (B) Comparing the influence of different number of rare codon insertions in luciferase production. Four Reporters are examined, including PT7-Luc-2AGG(BBa_K567008), PT7-Luc-4AGG(BBa_K567009), PT7-Luc-8AGG (BBa_K567010) and PT7-Luc-6AGG (BBa_K567019).

This picture reflects more clearly that the more rare codons are inserted, the lower the background expression and the narrower the range of device regulation. We are able to predict the outcome of influence of different number of rare codons in protein biosynthesis, offering valuable information for device usage.

Fig.4 The comparison between background expression and induced expression of luciferase with different rare codon insertions. (A)PT7-luc reporters. (B) Pbla-luc reporters

Influence of different strengths of target protein promoters

We examined the influence of different Reporter promoters on the working curve of our device, which is reflected by luciferase activity. The working range of our device is pre-defined by the strength of target protein promoter, T7 promoter and bla promoter in our project.

Fig.5 (A) Luciferase (Pbla-Luc-8AGG) production in cells overexpressing rare tRNA with lacI-Ptrc-tRNA^Arg. Luciferase production is reflected by bioluminescence emitted from the luciferin reaction. (B) Luciferase production in cells with PT7-Luc-8AGG as reporter. Here we analyze the influences of strong/weak promoter in luciferase production. Strong promoter (T7) of target gene can improve the titration curve, indicating that our device works better under strong target protein promoters.

Location of Rare Codons

Experiment results showed that when there is 111bp interval between the two-copy 4AGG, background is lower than there is a 30bp interval. In another analysis, protein production can be induced to a higher level when there are two copies of AGG tandems.

Fig.6 A. When there is 111bp interval between the two-copy 4AGG, background is lower than there is a 30bp interval. B. Protein production can be induced to a higher level when there are two copies of AGG tandems.

Experiment results showed that luciferase tagged with two-copy 4AGG insertions with an interval of 111 bp can increase yield and lower background noise of target protein. We further conducted experiments to test its characters.

When we compare the two-copy 4AGG tagged luciferase with the single copy one, we find that two-copy 4AGG tagged luciferase has a higher yield.

fig.7 Two-copy 4AGG tagged luciferase has a higher yield than single-copy

When we compare the two-copy 4AGG tagged luciferase with 8AGG tagged luciferase, we find that two-copy 4AGG tagged luciferase has a lower background.

fig.8 Two-copy 4AGG tagged luciferase has a lower background than 8AGG tagged luciferase

Related Biobrick

Pbla-Luc-2AGG (BBa_K567004)

Pbla-Luc-4AGG (BBa_K567005)

Pbla-Luc-6AGG (BBa_K567006)

Pbla-Luc-8AGG (BBa_K567007)

PT7-Luc-2AGG (BBa_K567008)

PT7-Luc-4AGG (BBa_K567009)

PT7-Luc-6AGG (BBa_K567019)

PT7-Luc-8AGG (BBa_K567010)

PT7-RFP-6AGG (BBa_K567017)

PT7-Luc-2x4AGG(111bp) (BBa_K567021)

PT7-Luc-2x4AGG(30bp) (BBa_K567026)

PT7-Luc-3x4AGG(30bp+111bp) (BBa_K567027)

Reference

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

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 1845
  
• 12
  INCOMPATIBLE WITH RFC[12]

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• 21
  COMPATIBLE WITH RFC[21]
• 23
  INCOMPATIBLE WITH RFC[23]

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• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 1845
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 2026