Composite

Part:BBa_K3883029

Designed by: Liu Yuqi   Group: iGEM21_Jilin_China   (2021-10-20)


Inhibitory effect of lacI on Ptrc

This part contains the trc promoter and the regulator gene lacI. By connecting the sfGFP gene downstream, the inhibitory effect of lacI on Ptrc is verified.

Usage and Biology

lacI, from Escherichia coli str.K-12, is a regulator gene in lac operon, located near the promoter lac P and encodes Lac repressor. Lac repressor proteins assemble into active tetramers that bind specifically to the manipulation site (O) in the lac operon and inhibit RNA polymerase recruitment, thereby inhibiting gene transcription. Ptrc, a heterozygous promoter constructed from Trp promoter and Lac promoter, is a strong promoter and can be inhibited by lac repressor. These two are used as part of our dynamic regulatory system.


Characterization

To verify the inhibitory effect of lacI on Ptrc, two genetic circuits were constructed (Fig1). The trends of fluorescence intensity were measured at the indicated time. As shown in the Fig1, the fluorescence of the experimental group was suppressed and stabilized at a low value, while that of the control group increased with time. It demonstrated that Ptrc is normally a strong constitutive promoter, while the Lac repressor encoded by lacI has a significant inhibitor on Ptrc.

Figure 1.Ptrc-sfGFP and lacI-Ptrc-sfGFP.



Figure 2.LacI can regulate downstream expression of Ptrc.](A). Digestion and gel electrophoresis of Ptrc-sfGFP. (B).The inhibitor of LacI on Ptrc. The plasmid was transformed into E.coli BL21(DE3). When the OD600 value of the bacteria reached 0.6, the fluorescence intensity of sfGFP was detected at the indicated time and normalized by the OD600 value. The experiment was performed three times in triplicate. *, P < 0.05 from respective control using Student’s test.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


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