Part:BBa_K4115006

LacI with lac promoter

Usage and Biology

Negative feedback control by lactose repressor (LacI) provides a classic example of bacterial gene expression regulation. In the absence of lactose, LacI blocks the transcription of genes encoding lactose-metabolizing enzymes by binding to a primary operator (O1) on the DNA, located just downstream of the RNA polymerase promoter. Transcription repression is strongly enhanced by the presence of two auxiliary operators, situated both upstream (O3) and downstream (O2) of the primary operator. This effect is due to the fact that LacI is a homotetrameric protein assembled as a dimer of dimers, with each dimer presenting a DNA-binding domain, enabling the tetramer to bind simultaneously to two operators and form a loop in the intervening DNA. Consequently, LacI binding to one of the auxiliary operators increases the proximity of the remaining free DNA-binding domain to the primary operator, thus enhancing the probability of transcription blocking.[1]

Figure 1: (A) LacI tetramer structure with DNA-binding domain in red, protein core in green and tetramerization domain in blue. Each dimer is shown in color tones of different darkness. (B) Different LacI-DNA loop topoisomers. Arrows indicate the 5′-3′ orientation of operators.[1]

In our project, the lacI gene is widely used for gene regulation in chassis prokaryotes. Under the regulation of the PlacI promoter, the normal expression of lacI protein inhibits the transcriptional activation ability of the lac UV5 promoter. When the IPTG inducer is added, the lacI protein binds to IPTG, the inhibitory effect stops, and the downstream genes of the lac UV5 promoter can be expressed normally.

Figure 2: Gene pathway map of the nifA-PnifH regulating system.

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]

[1]Rutkauskas, D., Zhan, H., Matthews, K. S., Pavone, F. S., & Vanzi, F. (2009). Tetramer opening in LacI-mediated DNA looping. Proceedings of the National Academy of Sciences of the United States of America, 106(39), 16627–16632.