Team:CityU HK/Experiments
Construction of a tightly regulated lactose inducible promoter
Building a tightly regulated lactose inducible promoter
This Biobrick is a designed for engineering a tightly regulated LacI inducible promoter. This Biobrick consists of three parts: the lacIQpromoter (PlacIQ), wild type lacI gene and the PL8-UV5 promoter, a modified glucose-independent LacI control promoter (Figure 1).
This Biobrick is a designed for engineering a tightly regulated LacI inducible promoter. This Biobrick consists of three parts: the lacIQpromoter (PlacIQ), wild type lacI gene and the PL8-UV5 promoter, a modified glucose-independent LacI control promoter (Figure 1).
Figure 1. Gene construct of BBa_K1695000. The biobrick consist three parts, the PlacIQ promoter, E. coli wild type lacI gene linked with RBS (BBa_B0034) and the LacI regulated promoter L8-UV5 .
PlacI Q
The PlacIQ is a mutated promoter of the lacI gene with a C --> T conversion in the -35 region (Calos, 1978) (Figure 2). According to Calos (1978), the LacI protein expression level is 10-fold higher in the PlacIQ system than the PlacI system.
Figure 2. The DNA sequences of the lacI and lacIQ promoters. Only the top strands of the promoters are depicted and the -10 and -35 sequences are shown in red boxes. The lacIQ mutation is highlighted in red.
Linking the constitutive promoter PlacIQ renders LacI being expressed constitutively and the extra LacI protein provides a stronger inhibition on the PL8-UV5 promoter.
PL8-UV5 (BBa_K1695000)
The PL8-UV5 promoter is a mutated lacI controlled promoter. The two single base-pair mutations (C --> T at positions -66 and -55) at the CAP-binding site inactivate the binding of CAP protein (Hirschel, Shen, & Schlessinger, 1980), leading to the promoter expression being independent of the cyclic AMP level, which are produced under poor glucose supply. In addition, a two-base pair mutation (GT --> AA at -9 and -8) converts the sequence at the -10 region back to the consensus sequence (TATAAT), which allows the σ factor to bind to the -10 element without the help of CAP protein (Figure 3).
Figure 3. Sequence of wild type Lac promoter and L8-UV5 Lac promoter. The CAP-binding site is underlined, the -10 element is boxed, and the two LacI binding sites (O3, O1) are highlighted gray. The mutations in L8-UV5 Lac promoter and the -10 region are highlighted red.
The use of the PL8-UV5 promoter can remove the inhibitory effect of glucose on lactose induction of the promoter. The expression of the gene downstream of this promoter is thus solely dependent on lactose concentration.