Difference between revisions of "Team:CityU HK/Experiments"
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| − | <h2 class="wsite-content-title" style="text-align:left;"> | + | <h2 class="wsite-content-title" style="text-align:left;"><span style=""><span style="">Module Description</span></span><br /></h2> |
| − | <div class="paragraph" style="text-align:left;"></div> | + | <div class="paragraph" style="text-align:left;"><font size="3"><span "font-size:12.0pt;mso-bidi-font-size:="" 11.0pt;font-family:"calibri","sans-serif";mso-ascii-theme-font:minor-latin;="" mso-fareast-font-family:新細明體;mso-fareast-theme-font:minor-fareast;mso-hansi-theme-font:="" minor-latin;mso-bidi-font-family:"times="" roman";mso-bidi-theme-font:minor-bidi;="" mso-ansi-language:en-us;mso-fareast-language:zh-tw;mso-bidi-language:ar-sa"="" style="">In the belief that everyone can enjoy dairy products, our team engineered an <em style="">E. coli </em>strain to help relieve of those people with lactose intolerance. The bacteria carry two recombinant genes which can synthesize </span><span "font-size:12.0pt;mso-bidi-font-size:11.0pt;font-family:symbol;="" mso-fareast-font-family:新細明體;mso-fareast-theme-font:minor-fareast;mso-bidi-font-family:="" "times="" roman";mso-bidi-theme-font:minor-bidi;mso-ansi-language:en-us;="" mso-fareast-language:zh-tw;mso-bidi-language:ar-sa"="" style="">beta</span><span "font-size:12.0pt;mso-bidi-font-size:11.0pt;font-family:"calibri","sans-serif";="" mso-ascii-theme-font:minor-latin;mso-fareast-font-family:新細明體;mso-fareast-theme-font:="" minor-fareast;mso-hansi-theme-font:minor-latin;mso-bidi-font-family:"times="" roman";="" mso-bidi-theme-font:minor-bidi;mso-ansi-language:en-us;mso-fareast-language:="" zh-tw;mso-bidi-language:ar-sa"="" style="">-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.</span></font><br /></div> |
| − | <h2 class="wsite-content-title" style="text-align:left;"> | + | <h2 class="wsite-content-title" style="text-align:left;"><font size="5">Lysis plasmid</font></h2> |
| − | <div class="paragraph" style="text-align:left;"></div></div> | + | <div><div class="wsite-image wsite-image-border-none " style="padding-top:10px;padding-bottom:10px;margin-left:0;margin-right:0;text-align:center"> |
| + | <a> | ||
| + | <img src="http://cityuigem2015.weebly.com/uploads/5/7/0/0/57008745/7119761.png?636" alt="Picture" style="width:636;max-width:100%" /> | ||
| + | </a> | ||
| + | <div style="display:block;font-size:90%">Fig2. The design of lysis plasmid</div> | ||
| + | </div></div> | ||
| + | |||
| + | <div class="paragraph" style="text-align:left;"><font size="3"><span style="">The lysis plasmid mainly is consisted of two parts: the lysis cassette and Lac repressor gene. </span><br /><span style=""></span><br /><span style=""></span> <span "font-size:12.0pt;mso-bidi-font-size:11.0pt;font-family:="" "calibri","sans-serif";mso-ascii-theme-font:minor-latin;mso-fareast-font-family:="" 新細明體;mso-fareast-theme-font:minor-fareast;mso-hansi-theme-font:minor-latin;="" mso-bidi-font-family:"times="" roman";mso-bidi-theme-font:minor-bidi;="" mso-ansi-language:en-us;mso-fareast-language:zh-tw;mso-bidi-language:ar-sa"="" style="">The lysis cassette is composed of holin (<em style="">S</em> gene), endolysin (<em style="">R</em> gene) and spanin (<em style="">Rz</em> 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 <em style="">S </em>gene were included. A missense mutation and a deletion of the trans-membrane domain were applied to the <em style="">S</em> 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.</span></font></div> | ||
| + | |||
| + | <div><div style="height: 30px; overflow: hidden; width: 100%;"></div> | ||
| + | <hr class="styled-hr" style="width:100%;"></hr> | ||
| + | <div style="height: 30px; overflow: hidden; width: 100%;"></div></div> | ||
| + | |||
| + | <h2 class="wsite-content-title" style="text-align:left;"><span "font-size:12.0pt;mso-bidi-font-size:11.0pt;font-family:"calibri","sans-serif";="" mso-ascii-theme-font:minor-latin;mso-fareast-font-family:新細明體;mso-fareast-theme-font:="" minor-fareast;mso-hansi-theme-font:minor-latin;mso-bidi-font-family:"times="" roman";="" mso-bidi-theme-font:minor-bidi;mso-ansi-language:en-us;mso-fareast-language:="" zh-tw;mso-bidi-language:ar-sa"="" style="">Characterization of the Biobricks/ parts</span></h2> | ||
| + | |||
| + | <h2 class="wsite-content-title" style="text-align:left;"><span "font-size:12.0pt;mso-bidi-font-size:11.0pt;font-family:"calibri","sans-serif";="" mso-ascii-theme-font:minor-latin;mso-fareast-font-family:新細明體;mso-fareast-theme-font:="" minor-fareast;mso-hansi-theme-font:minor-latin;mso-bidi-font-family:"times="" roman";="" mso-bidi-theme-font:minor-bidi;mso-ansi-language:en-us;mso-fareast-language:="" zh-tw;mso-bidi-language:ar-sa"=""></span><span "font-size:12.0pt;mso-bidi-font-size:11.0pt;font-family:"calibri","sans-serif";="" mso-ascii-theme-font:minor-latin;mso-fareast-font-family:新細明體;mso-fareast-theme-font:="" minor-fareast;mso-hansi-theme-font:minor-latin;mso-bidi-font-family:"times="" roman";="" mso-bidi-theme-font:minor-bidi;mso-ansi-language:en-us;mso-fareast-language:="" zh-tw;mso-bidi-language:ar-sa"="" style=""><font size="5">RNA levels of lacZ and lacY</font><br /></span></h2> | ||
| + | |||
| + | <div class="paragraph" style="text-align:left;"><font size="3">To determine the levels of the <em style=""><span style="">lacZ </span></em>and <em style=""><span style="">lacY</span></em> 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.<em style=""><span style=""></span></em><br /><br /> <strong>RNA extraction --> Reverse transcription --> Real time PCR</strong><br /> <br /><br /> <strong>RNA extraction:</strong> (Link to the protocol)<br /> To extract the total RNA from the engineered <em style="">E. coli</em><br /><br /> <em style=""><span style=""> </span></em><br /> <strong>Reverse transcription:</strong> (Link to the protocol)<br /> 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.<br /><br /> <br /> <strong>Real time PCR:</strong> (Link to the protocol)<br /> <span "font-size:12.0pt;mso-bidi-font-size:11.0pt;font-family:="" "calibri","sans-serif";mso-ascii-theme-font:minor-latin;mso-fareast-font-family:="" 新細明體;mso-fareast-theme-font:minor-fareast;mso-hansi-theme-font:minor-latin;="" mso-bidi-font-family:"times="" roman";mso-bidi-theme-font:minor-bidi;="" mso-ansi-language:en-us;mso-fareast-language:zh-tw;mso-bidi-language:ar-sa"="" style="">To amplify our desired cDNA (</span><em style=""><span "font-size:12.0pt;mso-bidi-font-size:11.0pt;font-family:"calibri","sans-serif";="" mso-ascii-theme-font:minor-latin;mso-fareast-font-family:新細明體;mso-fareast-theme-font:="" minor-fareast;mso-hansi-theme-font:minor-latin;mso-bidi-font-family:"times="" roman";="" mso-bidi-theme-font:minor-bidi;mso-ansi-language:en-hk;mso-fareast-language:="" zh-tw;mso-bidi-language:ar-sa"="" style="">lacZ</span></em><span "font-size:12.0pt;mso-bidi-font-size:11.0pt;font-family:"calibri","sans-serif";="" mso-ascii-theme-font:minor-latin;mso-fareast-font-family:新細明體;mso-fareast-theme-font:="" minor-fareast;mso-hansi-theme-font:minor-latin;mso-bidi-font-family:"times="" roman";="" mso-bidi-theme-font:minor-bidi;mso-ansi-language:en-us;mso-fareast-language:="" zh-tw;mso-bidi-language:ar-sa"="" style=""> 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.</span></font></div> | ||
| + | |||
| + | <h2 class="wsite-content-title" style="text-align:left;"><span "font-size:12.0pt;mso-bidi-font-size:11.0pt;font-family:"calibri","sans-serif";="" mso-ascii-theme-font:minor-latin;mso-fareast-font-family:新細明體;mso-fareast-theme-font:="" minor-fareast;mso-hansi-theme-font:minor-latin;mso-bidi-font-family:"times="" roman";="" mso-bidi-theme-font:minor-bidi;mso-ansi-language:en-us;mso-fareast-language:="" zh-tw;mso-bidi-language:ar-sa"=""><font size="5">Characterization on LacZ</font></span></h2> | ||
| + | |||
| + | <div class="paragraph" style="text-align:left;"><font size="3"><span style="">To determine the level of beta-galactosidase encoded by <em style="">lacZ</em>, ONPG assay was performed.</span><br /><br /><span style=""></span> <span style=""><strong>ONPG assay:</strong></span><span style=""> (link to the protocol)</span><br /><span style=""></span><br /><span style=""></span> <span style="">Besides lactose, ONPG (ortho-Nitrophenyl-β-galactoside<strong style="">)</strong> 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.</span></font><br /><span style=""></span><br /><span style=""></span></div> | ||
| + | |||
| + | <h2 class="wsite-content-title" style="text-align:left;"><span "font-size:12.0pt;mso-bidi-font-size:11.0pt;font-family:"calibri","sans-serif";="" mso-ascii-theme-font:minor-latin;mso-fareast-font-family:新細明體;mso-fareast-theme-font:="" minor-fareast;mso-hansi-theme-font:minor-latin;mso-bidi-font-family:"times="" roman";="" mso-bidi-theme-font:minor-bidi;mso-ansi-language:en-us;mso-fareast-language:="" zh-tw;mso-bidi-language:ar-sa"=""><font size="5">pL8-UV5 characterization</font></span></h2> | ||
| + | |||
| + | <div class="paragraph" style="text-align:left;"><span style=""><font size="3">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.</font></span><br /><span style=""></span><br /><span style=""></span></div> | ||
| + | |||
| + | <h2 class="wsite-content-title" style="text-align:left;"><font size="5">Lysis cassette characterization</font><br /><span style=""></span></h2> | ||
| + | |||
| + | <div class="paragraph" style="text-align:left;"><font size="3"><span style="">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. </span><br /><br /><span style=""></span> <span style="">The O.D. and the colony forming units (CFU) were measured to determine the efficiency of the lysis cassette. </span><br /><span style=""></span><br /><span style=""></span> <span style="">Inducing the promoter with lactose --> </span><span style="">Measure O.D. --> </span><span style="">Serial dilution --> </span><span style="">Spread plate</span></font><span style=""> --> </span><font size="3"><span style="">Incubate</span></font><span style=""> --> </span><font size="3"><span style="">Count the number of colonies</span><br /><span style=""></span><br /><span style=""></span> <span style="">The lower the CFU/O.D., the higher the efficiency of the lysis cassette after induction</span><br /></font><span style=""></span><br /><span style=""></span></div> | ||
| + | |||
| + | <h2 class="wsite-content-title" style="text-align:left;"><span "font-size:12.0pt;mso-bidi-font-size:11.0pt;font-family:"calibri","sans-serif";="" mso-ascii-theme-font:minor-latin;mso-fareast-font-family:新細明體;mso-fareast-theme-font:="" minor-fareast;mso-hansi-theme-font:minor-latin;mso-bidi-font-family:"times="" roman";="" mso-bidi-theme-font:minor-bidi;mso-ansi-language:en-us;mso-fareast-language:="" zh-tw;mso-bidi-language:ar-sa"=""><font size="5">Characterization of the lacY lacZ operon BBa_S04055 (from 2008 Caltech: Curing lactose intolerance)</font></span></h2> | ||
| + | |||
| + | <div class="paragraph" style="text-align:left;"><span "font-size:12.0pt;mso-bidi-font-size:="" 11.0pt;font-family:"calibri","sans-serif";mso-ascii-theme-font:minor-latin;="" mso-fareast-font-family:新細明體;mso-fareast-theme-font:minor-fareast;mso-hansi-theme-font:="" minor-latin;mso-bidi-font-family:"times="" roman";mso-bidi-theme-font:minor-bidi;="" mso-ansi-language:en-us;mso-fareast-language:zh-tw;mso-bidi-language:ar-sa"="" style=""><font size="3">Western blotting (link to the protocol) is used to detect the expression level of beta-galactosidase inside <em style="">E. coli</em> harboring BBa_S04055.</font></span></div></div> | ||
</div> | </div> | ||
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Revision as of 00:14, 18 September 2015
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 beta-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.
Lysis plasmid
Fig2. 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 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.