Part:BBa_K567004

Pbla-Luc-2AGG

β-lactamase promoter-Luciferase with two AGG-codon insertions. This biobrick is constructed by putting modified enzyme luciferase under constituitive promoter β-lactamase promoter. 2 AGG codons and 2 GCG codons are inserted after the ATG start codon of wild type luciferase (BBa_I712019). Modified luciferase keeps the activity of converting luciferin into oxyluciferin, during which bioluminescence will emit. This part is one of the reporter genes to testify the influence of different number of rare codons in regulating protein biosynthesis. This part is used as a measurement to testify the function of LacI -Ptrc-tRNA(Arg)(BBa_K567001) or sulA promoter-tRNA(Arg) (BBa_K567002). Cell is cultured in 50ug/ml kanamycin and 10ug/ml tetracycline LB liquid medium. When the OD600 of the culture reaches 0.3 IPTG is added to make the final concentration 0.5nM to induce the synthesis of tRNA. Ultrasonication is used to release the luciferase from the cell. Sonics ON 3 seconds, OFF 3 seconds, total ultrasonication time 3minutes. Amount of bioluminescence produced can be detected using luminometer.

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.1 (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.2 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.3 (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.

Get more information from Biobrick lacI-Ptrc-tRNA^Arg (BBa_K567001)

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 1100