Coding

Part:BBa_K3257045

Designed by: Yi Qiao   Group: iGEM19_Fudan-TSI   (2019-09-05)
Revision as of 11:06, 10 October 2019 by YY12138 (Talk | contribs)

Improved LacI

This year team Fudan-TSI has upgraded LacI gene (BBa_C0012 https://parts.igem.org/Part:BBa_K3257012) to a better version.

LacI is one of the genes in Lac operon encoding the inhibitor protein binding to LacO sites (cis-acting element). In response to IPTG, the inhibitor protein detaches from LacO (BBa_K3257066 https://parts.igem.org/Part:BBa_K3257066) and enables the transcription of downstream genes. We mutated some specific sites in the LacI gene to improve its sensibility to IPTG.[1] Using EGFP (BBa_E0040 https://parts.igem.org/Part:BBa_E0040) as a reporter, its fluorescence intensity appears a lower leakage and the same level of expression before and after the induction of IPTG. Also, we induce the improved Lac operon by arabinose to verify its orthogonal response to IPTG.

With lacIq promoter (BBa_K3257003 https://parts.igem.org/Part:BBa_K3257003) and rrnB T1 terminator (BBa_K3257020 https://parts.igem.org/Part:BBa_K3257020), improved LacI protein can be expressed and function properly in the Escherichia coli BL21(DE3). We used EGFP as a reporter controlled by our improved Lac operon and measured its green fluorescence over time.

According to our experiment, our Lac operon is improved in the following three main aspects.

Lower Response to Arabinose, Better Orthogonality

Cross talk between the response to IPTG and arabinose has been a defect of the wild type Lac operon. When 4mM arabinose added, a few lac inhibitors detach from lac operator which means that it is induced in a relatively low but unignorable level. According to the measurement of our experiment, our improved LacI can respond to IPTG with better orthogonality. The figure below (Figure 1) is the measurement of the fluorescence of EGFP controlled by wild-type and improved Lac operon. It shows that when 4mM arabinose is added, oLacI(wild-type LacI) is induced at a significantly high level while iLacI(improved LacI) is induced at a lower level.

Flow cytometry results of the original published SynNotch. surEGFP is the surface expressed EGFP, which was used as the antigen. Without antigen (+Mock), the EGFP (Y axis, driven by tTAA released after SynNotch activation) was low, and it went "high" after adding the antigen. More details please visit http://2018.igem.org/Team:Fudan/Results and http://2018.igem.org/Team:Fudan/Optimization .

Same Level of Expression as the Wild-type Lac operon when Induced by IPTG

Next step we prove that our improved Lac operon can be normally induced by IPTG, which counts a great deal for the usage of this operon. The figure below (Figure 2) is the measurement of the fluorescence of EGFP controlled by wild-type and improved Lac operon. It shows that when 1mM IPTG is added, EGFP controlled by both operons can be disinhibited and expressed at a relatively same level.

Lower Uninduced Leakage

The uninduced leakage level is also an important parameter of an operon. Improved LacI lowers the leakage level compared to the wild-type one. The figure below (Figure 3) is the measurement of the fluorescence of EGFP controlled by wild-type and improved Lac operon. When no IPTG or arabinose is added, the fluorescence of EGFP controlled by improved Lac operon is under the detection range while the fluorescence of EGFP controlled by wild-type Lac operon remains a detectable signal indicating a considerably undesired leakage.


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
    COMPATIBLE WITH RFC[1000]


Reference

[1] AJ Meyer et al. Escherichia coli "Marionette" strains with 12 highly optimized small-molecule sensors. Nat Chem Biol. 2019 Feb;15(2):196-204. doi: 10.1038/s41589-018-0168-3. Epub 2018 Nov 26.


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