Regulatory

Part:BBa_K1695000

Designed by: Aruna Surendran Ramkrishnan   Group: iGEM15_CityU_HK   (2015-08-10)

Tightly-regulated lactose-inducible promoter

This Biobrick is designed to bioengineer a tightly regulated lactose-inducible expression system. There are 3 components in this Biobrick: a lacIQ promoter (PlacIQ), a wild-type lacI gene and the PL8-UV5 promoter (consisting of a modified glucose-independent LacI-regulated promoter sequence) (Figure 1).

2015CityU HK PL8UV5.png

Figure 1. Lactose-inducible promoter construct (BBa_K1695000). This biobrick consists of three parts: the PlacIQ promoter, wild-type lacI gene with a RBS (BBa_B0034), and the LacI-regulated promoter L8-UV5.


PlacIQ

PlacIQ is a mutated promoter of the lacI gene which has a C -> T change in the -35 region (Figure 2). According to Calos (1978), expression of the LacI protein is 10-fold higher in the PlacIQ system than the wild type PlacI system.

2015CityU HK lacIlacIQ.png

Figure 2. Single nucleotide difference between the lacI and lacIQ promoters. The upper strand of either promoters are depicted, and the -10 and -35 sequences are highlighted in red boxes. The T mutation in lacIQ is highlighted in red.

Adding the constitutive promoter PlacIQ to the construct renders LacI proteins being expressed constitutively and the extra LacI protein in turn exerting stronger inhibition on the PL8-UV5 promoter.

PL8-UV5

PL8-UV5 is a mutated lacI-regulated promoter. There are two base mutations (C -> T at positions -66 and -55) in the CAP-binding site, which prevent binding of the CAP protein (Hirschel et al., 1980), resulting in the promoter being non-responsive to cyclic AMP levels (which are produced at high levels under low glucose conditions). In addition, a two-base mutation (GT -> AA) at the ‒9 and ‒8 nucleotide positions, converts the sequence at the -10 region back to the consensus TATAAT sequence which allows the σ factor to bind to the -10 element without the help of a CAP protein (Figure 3). Addition of the PL8-UV5 promoter to the biobrick is predicted to remove the inhibitory effect of glucose on lactose induction of the promoter. As a consequence, expression of any gene(s) downstream of this promoter will be tightly regulated and dependent on lactose concentration.

2015CityU HK PL8UV5 figure 3.png

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.

Characterization of the lactose-inducible promoter (BBa_K1695053)

The GFP reporter biobrick (BBa_K1695053) was constructed by linking the above lacI-regulated promoter (BBa_K1695000) to GFP (BBa_I13504) (Figure 4). The response of this GFP reporter construct to IPTG induction was then measured based on the GFP fluorescence signal (excitation wavelength: 485nm; emission wavelength: 520 nm). GFP fluorescence signal from cells carrying the PL8-UV5-GFP construct (BBa_K1695053) was monitored over a 4.5-hour time course under a range of IPTG concentrations ranging from 0.01 – 10 mM. Normalization of GFP fluorescence was performed based on the A600 values of the culture.

2015CityU HK BBa K1695053.png

Figure 4. Construct of the reporter biobrick BBa_K1695053


(A)2015CityU HK PL8UV5 Char1.png

(B)2015CityU HK PL8UV5 Char2.png

Figure 5. Expression analysis of L8-UV5 promoter-GFP biobrick (BBa_K1695053). (A) Activation of GFP expression in the L8-UV5 promoter construct is dependent on IPTG concentrations below 1 mM. (B) Fluorescence response of the L8-UV5 promoter-GFP gene construct (BBa_K1695053) was measured over a 4.5-h time course under a range of IPTG concentrations (from 0.01 mM to 10 mM). Results show that induction of the L8-UV5 promoter is independent of IPTG at concentrations above 1 mM, where LacI is saturated and promoter is fully turned on.

Results in Figure 5 show that GFP expression in the L8-UV5 promoter-GFP construct is dependent on IPTG concentrations below 1 mM. At IPTG concentrations above 1 mM, expression of GFP has reached a maxima regardless of the sampling time points (Figure 5).



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]


[edit]
Categories
Parameters
None