Difference between revisions of "Team:CityU HK/Experiments"
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<h2 class="wsite-content-title" style="text-align:left;"><font size="5"><b>P<em style="">lacI </em><sup>Q</sup></b></font></h2> | <h2 class="wsite-content-title" style="text-align:left;"><font size="5"><b>P<em style="">lacI </em><sup>Q</sup></b></font></h2> | ||
| − | <div class="paragraph" style="text-align:left;"><font size="3"><span style="">The P<em style="">lacI</em><sup>Q</sup> is a mutated promoter of the <em style="">lacI</em> 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 P<em style="">lacI</em><sup>Q</sup> system than the P<em style="">lacI</em> system.</span></font></div> | + | <div class="paragraph" style="text-align:left;"><font size="3"><span style="">The P<em style="">lacI</em><sup>Q</sup> is a mutated promoter of the <em style="">lacI</em> 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 P<em style="">lacI</em><sup>Q</sup> system than the P<em style="">lacI</em> system.</span></font><br /><br /></div> |
| + | |||
| + | <div class="paragraph" style="text-align:left;"><font size="3"><span style="">Linking the constitutive promoter P<em style="">lacI</em><sup>Q</sup> renders LacI being expressed constitutively and the extra LacI protein provides a stronger inhibition on the PL8-UV5 promoter.</span></font></div> | ||
<div><div style="height: 30px; overflow: hidden; width: 100%;"></div> | <div><div style="height: 30px; overflow: hidden; width: 100%;"></div> | ||
Revision as of 04:24, 18 September 2015
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.
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.