Team:CityU HK/Experiments
Module Description
In the belief that everyone can enjoy dairy products, our team engineered an E. coli strain to help relieve of those people with lactose intolerance. The bacteria carry two recombinant genes which can synthesize β-galactosidase, the enzyme for breaking down lactose and lactose permease, allowing lactose to enter the bacteria faster. Lysis plasmid was constructed with features to allow the release of lactase quickly once the bacteria sense the presence of lactose in the surrounding.
A. lacZY plasmid
Figure 1. The design of lacZY plasmid .
The lacZ gene encodes β-galactosidase, which is an enzyme that digests lactose into glucose and galactose. A strong ribosome binding site (BBa_B0034) is added in front of the lacZ gene to increase the binding affinity of ribosomes to RBS. The lacY’ gene, preceded by a weak ribosome binding site (BBa_B0033), encodes for a mutated lactose permease that cannot be inhibited by glucose, and allows efficient transport of lactose into the cells. The constitutive promoter BBa_J23100 provides a constant and strong transcription for the two genes, which ensue sufficient production of β-galactosidase and allows transport of lactose for activating lysis plasmid.
B. 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 2).
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 2).
Figure 2. 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 3. 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 4).
Figure 4. 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.
C. Lysis plasmid
Figure5. The design of lysis plasmid
The lysis plasmid mainly is consisted 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 β-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 9 lysis cassettes with different combinations (please refer to the PARTS). 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 β-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 9 lysis cassettes with different combinations (please refer to the PARTS). 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 β-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 β-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 β-galatosidase was determined.
ONPG assay: (link to the protocol)
Besides lactose, ONPG (ortho-Nitrophenyl-β-galactoside) is also a substrate of β-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 β-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 β-galactosidase inside E. coli harboring BBa_S04055.