Part:BBa_K567021

PT7-Luc-2x4AGG(111bp)

This biobrick is constructed by putting modified enzyme luciferase under promoter T7 and controlled by lac operator. 4 AGG codons and 2 GCG codons are inserted in TWO DIFFERENT places 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 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.

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.4 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.5 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.6 Two-copy 4AGG tagged luciferase has a lower background than 8AGG tagged luciferase

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

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal XbaI site found at 48
  
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal XbaI site found at 48
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal XbaI site found at 48
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 1040