Composite

Part:BBa_K567001

Designed by: Chunying Li and Yunfeng Ruan   Group: iGEM11_SJTU-BioX-Shanghai   (2011-09-29)
Revision as of 03:27, 29 October 2011 by Alfred (Talk | contribs)

lacI-Ptrc-tRNA(Arg)

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

Design

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

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

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

Over expressed tRNA(Arg)-AGG is charged by native Arginyl-tRNA Synthetase(ArgRS)

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

6AGG codons are inserted after the start codon ATG in the reporter gene

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.

11SJTU rare 01.jpg



Result

Fig.1 Confocal Microscope examining RFP expression. RFP has been largely produced in cells overexpressing tRNAArg-AGG.
Fig.2 Cells over-expressing tRNAArg-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 tRNAArg. No RFP can be observed in cells without rare tRNA overexpression.

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

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


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)


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 1845
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 1845
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 1845
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 1845
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 2026


[edit]
Categories
Parameters
chassisEscherichia coli
functionIPTG induced tRNA expression
ligandsIsopropyl β-D-1-Thiogalactopyranoside
n/alacI-Ptrc-tRNA(Arg)