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 lacI Qpromoter (PlacI Q), 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 lacI Qpromoter (PlacI Q), wild type lacI gene and the PL8-UV5 promoter, a modified glucose-independent LacI control promoter (Figure 1).
Lysis plasmid
Figure 1. The design of lysis plasmid
The lysis plasmid mainly consists of two parts: the lysis cassette and Lac repressor gene.
The lysis cassette is composed of holin (S gene), endolysin (R gene) and spanin (Rz gene). Two phage origins of the lysis cassette, phage lambda and phage 21, were cloned and compared. To speed up the cell lysis for releasing beta-galactosidase, 2 modifications including codon optimization and mutations on the S gene were included. A missense mutation and a deletion of the trans-membrane domain were applied to the S gene of phage lambda and phage 21, respectively. Our team has constructed 10 lysis cassettes with different combinations (Table1). For all the cassettes, pL8-UV5, a LacI regulated promoter was used to turn the transcription on.
The lysis cassette is composed of holin (S gene), endolysin (R gene) and spanin (Rz gene). Two phage origins of the lysis cassette, phage lambda and phage 21, were cloned and compared. To speed up the cell lysis for releasing beta-galactosidase, 2 modifications including codon optimization and mutations on the S gene were included. A missense mutation and a deletion of the trans-membrane domain were applied to the S gene of phage lambda and phage 21, respectively. Our team has constructed 10 lysis cassettes with different combinations (Table1). For all the cassettes, pL8-UV5, a LacI regulated promoter was used to turn the transcription on.
Characterization of the Biobricks/ parts
RNA levels of lacZ and lacY
To determine the levels of the lacZ and lacY transcripts, total RNA extraction was extracted, RNA was converted into cDNA by reverse transcription and the levels of the two transcripts were determined by real time PCR.
RNA extraction --> Reverse transcription --> Real time PCR
RNA extraction: (Link to the protocol)
To extract the total RNA from the engineered E. coli
Reverse transcription: (Link to the protocol)
To convert the total RNA into cDNA for the subsequent real time PCR. Real time PCR can only quantify the levels of cDNA but not RNA.
Real time PCR: (Link to the protocol)
To amplify our desired cDNA (lacZ and lacY) if the RNA extract contains lacZlacY RNA. It will be difficult to detect the small amount of RNA in cells if the RNA, which is then converted to cDNA), is not amplified.
RNA extraction --> Reverse transcription --> Real time PCR
RNA extraction: (Link to the protocol)
To extract the total RNA from the engineered E. coli
Reverse transcription: (Link to the protocol)
To convert the total RNA into cDNA for the subsequent real time PCR. Real time PCR can only quantify the levels of cDNA but not RNA.
Real time PCR: (Link to the protocol)
To amplify our desired cDNA (lacZ and lacY) if the RNA extract contains lacZlacY RNA. It will be difficult to detect the small amount of RNA in cells if the RNA, which is then converted to cDNA), is not amplified.
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.