Part:BBa_K3505003

LacI-Lac repressor GB compatible with B3-B5

Coding region for the LacI protein without an RBS.

Usage and Biology

The lac inhibitor and its operator is usually use for gene regulation and together they form a genetic circuit which called : the lac operon. In bacteria ,in abundance of lactose , lactose binds to lacI and the lac operon is activated. In synthetic biology , as an inducer we use IPTG, which doesn’t degraded in the cell , instead of lactose . IPTG is a molecular mimic of a lactose metabolite, which activates transcription of the lac operon. The lacI has the ability to bind to the lac operator (BBa_R0010 and BBa_R0011 hybrid regulator) inhibiting the expression of the desirable transcription unit.

Design Notes

The coding sequence was domesticated . We removed BsmBI ,BsaI , BtgZI, BpiI sites in order to be compatible with GoldenBraid and MoClo. The sequence is presents in pUPD2 and has overhangs compatible for Golden Braid cloning. The CDS has position B3-B5.

Figure 1. Enzymatic assay of β-lactamase with nitrocefin as its substrate, when expressed from a non-regulated and a toehold-regulated construct in a cell-free system. Error bars correspond to the standard deviation of n=2 replicates. Blank was subtracted. After the 3-hour incubation in the cell-free system, the chromogenic substrate of β-lactamase, nitrocefin, was added and an additional 45-minute enzymatic assay was performed in the plate reader, at 37 ℃.

The pre-existing part BBa_C0012has a C-terminal peptide that mediates degradation in bacteria (LVA tail). Our sequence doesn't contain LVA tail.

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]

Source

E.Coli , sequence found through Addgene https://www.addgene.org/82021/

References

• Lewis M. The lac repressor. C R Biol. 2005 Jun;328(6):521-48. doi: 10.1016/j.crvi.2005.04.004. PMID: 15950160.
• Elowitz, M.B., Leibler, S. A synthetic oscillatory network of transcriptional regulators. Nature 403, 335-338 (2000).