Team:CityU HK/Experiments
BBa_K1695000
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.
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
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.
Characterization on LacZ
To determine the level of beta-galactosidase encoded by lacZ, ONPG assay was performed.
ONPG assay: (link to the protocol)
Besides lactose, ONPG (ortho-Nitrophenyl-β-galactoside) is also a substrate of beta-galactosidase. ONPG is digested into galactose and ortho-nitrophenol (ONP) which is yellow in colour. Chloroform was used to lyse the cells and the enzymes were released into the surrounding. By measuring the O.D. at 420 nm after lysing the cells, the level of the beta-galatosidase was determined.
ONPG assay: (link to the protocol)
Besides lactose, ONPG (ortho-Nitrophenyl-β-galactoside) is also a substrate of beta-galactosidase. ONPG is digested into galactose and ortho-nitrophenol (ONP) which is yellow in colour. Chloroform was used to lyse the cells and the enzymes were released into the surrounding. By measuring the O.D. at 420 nm after lysing the cells, the level of the beta-galatosidase was determined.
pL8-UV5 characterization
pL8-UV5 is a regulated promoter which can be induced by either lactose or the analog IPTG. To characterize the inducible property of the pL8-UV5 promoter, pL8-UV5 was engineered upstream of the GFP gene. The GFP will be transcribed and translated after the promoter is induced. GFP fluorescent signal will be given by GFP. By measuring the GFP fluorescent signal after adding different concentration of IPTG to the bacteria, the inducible property of pL8-UV5 promoter can be determined.
Lysis cassette characterization
To characterize the efficiency of the lysis cassette, the lysis cassette was put under the control of theT7 promoter in the pSNAP plasmid. By inducing the T7 promoter with lactose, the genes in the lysis cassette was transcribed and translated into the components of the lysis proteins, which cause cell lysis.
The O.D. and the colony forming units (CFU) were measured to determine the efficiency of the lysis cassette.
Inducing the promoter with lactose --> Measure O.D. --> Serial dilution --> Spread plate --> Incubate --> Count the number of colonies
The lower the CFU/O.D., the higher the efficiency of the lysis cassette after induction
The O.D. and the colony forming units (CFU) were measured to determine the efficiency of the lysis cassette.
Inducing the promoter with lactose --> Measure O.D. --> Serial dilution --> Spread plate --> Incubate --> Count the number of colonies
The lower the CFU/O.D., the higher the efficiency of the lysis cassette after induction
Characterization of the lacY lacZ operon BBa_S04055 (from 2008 Caltech: Curing lactose intolerance)
Western blotting (link to the protocol) is used to detect the expression level of beta-galactosidase inside E. coli harboring BBa_S04055.