Part:BBa_K3505003

LacI-Lac repressor GB compatible with B3-B5

Coding region for the LacI protein without an RBS. The pre-existing part BBa_C0012has a C-terminal peptide that mediates degradation in bacteria (LVA tail). Our sequence doesn't contain LVA tail.

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 don't degrade 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.The overhangs of this part in the GoldenBraid Grammar.

Figure 2. The level 0 module : pupd2- LacI (illustration from SnapGene)

Verification of Cloning

Fig.3:(U=Uncut C=Cut) Restriction Digestion of LacI with EcoRI + SlaI, Expected bands : 3189 +2300 + 892 bp, Positive Result:C2

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).