Reporter

Part:BBa_K2276010

Designed by: Tianying Chen   Group: iGEM17_SCU_China   (2017-10-24)
Revision as of 23:19, 1 November 2017 by Greeny (Talk | contribs) (→‎Measurement)


a composite of TetR repressible promoter、special designed RBS and the coding sequence of mRFP

The part was designed based on the Part:BBa_I13521,while the RBS binding site is modified. The RBS is special designed for strainS whose last 9 nucleotides of the 16S rRNA is ACCTCCTTA.And it can be regard as a improvement for it has a high translation rate compared with Part:BBa_I13521.The promoter is TetR repressible promoter which is also used in the repressilator system. And the mRFP served as the reporter to help us measure the translation rate of the designed RBS.

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
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 635
    Illegal AgeI site found at 747
  • 1000
    COMPATIBLE WITH RFC[1000]


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
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 634
    Illegal AgeI site found at 746
  • 1000
    COMPATIBLE WITH RFC[1000]


Measurement

In order to improve the expression of the original part:BBa_I13521, we predicted a synthetic RBS sequence, named P3, with much higher translation initiation rate via the RBS Calculator designed by H.M. Salis, and constructed this part BBa_K2276010 containing RBS P3. And then, we measured the fluorescence intensity and OD600 changes of it. As Fig 1 and Fig 2 showed,Part: the expression of BBa_K2276010 was much stronger than the original part: BBa_I13521. In addition, we calculated the relative RBS strength via dividing the ratio of fluorescence intensity and OD 600 by fluorescence intensity of B0034. And this result further illustrated that this part contained a obviously stronger RBS than BBa_B0034. Thus, the function of the original part BBa_I13521 was indeed improved by us.

fig.1The fluorescence intensity changes over time (0h~16h). P1 represent the strain containing part: BBa_K2276007. P2 represent the strain containing part: BBa_K2276008. P3 represent the strain containing part: BBa_K2276010. TetR represent the strain that only has tetR repressible promoter without RBS and protein coding sequence. B0034 represent the strain that containing Part: BBa_I13521.
fig.2 The OD600 changes over time(0~16 h).P1 represent the strain containing part: BBa_K2276007. P2 represent the strain containing part: BBa_K2276008. P3 represent the strain containing part: BBa_K2276010. TetR represent the strain that only has tetR repressible promoter without RBS and protein coding sequence. B0034 represent the strain that containing Part: BBa_I13521.
fig.3 The relative strength.P1 represent the strain containing part: BBa_K2276007. P2 represent the strain containing part: BBa_K2276008. P3 represent the strain containing part: BBa_K2276010. B0034 represent the strain that containing Part: BBa_I13521.

Source

BBa_I13521

References

[1] Borujeni A E, Salis H M. Translation Initiation is Controlled by RNA Folding Kinetics via a Ribosome Drafting Mechanism[J]. Journal of the American Chemical Society, 2016, 138(22):7016.

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