Team:GeorgiaTech/Parts

From 2014.igem.org

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<h1>Biobricks and Devices</h1>
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<th width="20%">Name</th><th width="20%">Type</th><th width="50%">Description</th><th width="10%">Length</th>
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<tr><td>BBa_K1539001<td>Part<td>Coding sequence has a high efficiency promoter followed by a high efficiency RBS to express fluorescent protein mCherry.<td>814 bp
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<tr><td>BBa_K1539002<td>Part<td>Coding sequence has a low efficiency promoter followed by a low efficiency RBS to express fluorescent protein mCherry.<td>814 bp
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<tr><td>BBa_K1539003<td>Part<td>This coding sequence is for the beta subunit for Protein A (composed of three subunits) of the multi-complex enzyme soluble methane monooxyegnase (sMMO). The gene has been codon optimized for E. coli.<td>1170 bp
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<tr><td>BBa_K1539005<td>Part<td>This coding sequence is for the gamma subunit for Protein A (composed of three subunits) of the multi-complex enzyme soluble methane monooxyegnase (sMMO). The gene has been codon optimized for E. coli.<td>513 bp
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<tr><td>BBa_K1539008<td>Part<td>This coding sequence is for the gene sMMO B, encoding Protein B of the multi-complex enzyme soluble methane monooxyegnase (sMMO). The gene has been codon optimized for E. coli.<td>426 bp
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<tr><td>BBa_K1539013<td>Part<td>This coding sequence is for the gene sMMO C, encoding Protein C of the multi-complex enzyme soluble methane monooxyegnase (sMMO). The gene has been codon optimized for E. coli.<td>1047 bp
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<tr><td>BBa_K1539021<td>Device<td>This primer is used to insert a high efficiency RBS to transcribe genes starting with ATG in E. coli.<td>46 bp
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<tr><td>BBa_K1539034<td>Device<td>This primer is used to insert a low efficiency RBS to transcribe genes starting with ATG in E. coli.<td>46 bp
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<tr><td>BBa_K1539055<td>Device<td>This primer is used to insert a high efficiency promoter to translate E. coli. genes. Must be used after insertion of RBS primer. Use Georgia Tech iGEM RBS primers (BBa_K1539021 or BBa_K1539034) or an RBS primer with a similar design.<td>58 bp
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<h4 style="font: 18px 'Bitter' serif;">Jump to:</h4>
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<tr><td>BBa_K1539089<td>Device<td>This primer is used to insert a low efficiency promoter to translate E. coli. genes. Must be used after insertion of RBS primer. Use Georgia Tech iGEM RBS primers (BBa_K1539021 or BBa_K1539034) or an RBS primer with a similar design.<td>58 bp
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    <a href="#May" style="font: 18px 'Bitter' serif; color: #B22222;">May</a>
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    <a href="#June" style="font: 18px 'Bitter' serif; color: #B22222;">June</a>
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    <a href="#July" style="font: 18px 'Bitter' serif; color: #B22222;">July</a>
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<h1 id="Notebook">Notebook</h1>
 
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<h3 id="May">May</h3>
 
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<h2>Project Design</h2>
 
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<p>The month of May was largely focused on the selection and design of our Biobrick parts and devices. Two weeks were dedicated to the selection process for our team's 2014 iGEM project. Each acting team member identified a problem and proposed a solution via a genetically engineered organism; through discussion and popular vote, it was eventually determined that our team would work to express functional sMMO in E.coli for the purpose of methane remediation.</p>
 
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<p>At this time, the team also voted to continue characterization and expansion of the RBS insertion primers first designed by Georgia Tech's 2013 iGEM Team.</p>
 
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<p>May saw very little bench work besides training modules to develop wet lab skills and build our supply of biological resources.</p>
 
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<h3 id="June">June</h3>
 
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<h2>RBS and Promoter Primer Testing</h2>
 
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<p>Starting in June the team began testing of newly designed RBS insertion primers as well as newly designed promoter insertion primers based upon the same concept. Initial tests with these primers ultimately failed. It was deemed that the protocols and techniques developed by the team were not adequate enough for job. However, by the end of the month the team had achieved successes in inserting both a high and a low efficiency RBS site in front of the mCherry reporter gene in pSB1C3.</p>
 
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<h2>sMMO Project Planning</h2>
 
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<p>No bench work was performed on the sMMO project in the month of June, instead the time was spent meticulously designing our selected sMMO genes for DNA synthesis in order to ensure optimized expression in E. coli and an easy to manipulate gene in the lab. Additionally, the high cost of synthetic DNA drove our team to spend a great deal of time fundraising and obtaining discounts from molecular biology product suppliers.</p>
 
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<h3 id="July">July</h3>
 
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<h2>RBS and Promoter Primer Function Verification</h2>
 
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<p>Come July, refinement of the techniques and protocols used for RBS and promoter insertion finally came to fruition. Breaking the protocol into separate steps for RBS insertion and promoter insertion as well as a re-designing of the original promoter primers led to the verification of three new RBS primers and two new promoter primers. Although the strength of these PCR inserted sites had not yet been checked by flow cytometry, sequencing showed that the sequences had been inserted as predicted.</p>
 
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<h2>sMMO Gene Arrival</h2>
 
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<p>The synthesized sMMO genes arrived during this month, and all efforts went towards inserting these genes into vectors (if they weren't already) and transforming E.coli with them for the purpose of creating safe frozen stocks.</p>
 
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<h3 id="August">August</h3>
 
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<h2>RBS and Promoter Relative Expression Analysis</h2>
 
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<p>Finally Flow cytometry characterization of the RBS and promoter primers was initiated. The cytometry provided information on the relative expression rates of combinations of various strength RBS and promoters. This knowledge aided in our selection of primers to use with the individual sMMO genes.</p> <p>It was noted that the value of these primers would be more tangible if they were also compared to an mCherry reporter with an RBS and promoter site synthesized through more traditional 3A assembly or ordered digestion and ligation.</p>
 
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<h2>Generation of New Biobrick Parts</h2>
 
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<p>sMMO genes were inserted into Biobrick format over the month of August in vector pSB1C3.</p>
 
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<h3 id="September">September</h3>
 
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Latest revision as of 05:26, 15 October 2014

Biobricks and Devices

NameTypeDescriptionLength
BBa_K1539001PartCoding sequence has a high efficiency promoter followed by a high efficiency RBS to express fluorescent protein mCherry.814 bp
BBa_K1539002PartCoding sequence has a low efficiency promoter followed by a low efficiency RBS to express fluorescent protein mCherry.814 bp
BBa_K1539003PartThis coding sequence is for the beta subunit for Protein A (composed of three subunits) of the multi-complex enzyme soluble methane monooxyegnase (sMMO). The gene has been codon optimized for E. coli.1170 bp
BBa_K1539005PartThis coding sequence is for the gamma subunit for Protein A (composed of three subunits) of the multi-complex enzyme soluble methane monooxyegnase (sMMO). The gene has been codon optimized for E. coli.513 bp
BBa_K1539008PartThis coding sequence is for the gene sMMO B, encoding Protein B of the multi-complex enzyme soluble methane monooxyegnase (sMMO). The gene has been codon optimized for E. coli.426 bp
BBa_K1539013PartThis coding sequence is for the gene sMMO C, encoding Protein C of the multi-complex enzyme soluble methane monooxyegnase (sMMO). The gene has been codon optimized for E. coli.1047 bp
BBa_K1539021DeviceThis primer is used to insert a high efficiency RBS to transcribe genes starting with ATG in E. coli.46 bp
BBa_K1539034DeviceThis primer is used to insert a low efficiency RBS to transcribe genes starting with ATG in E. coli.46 bp
BBa_K1539055DeviceThis primer is used to insert a high efficiency promoter to translate E. coli. genes. Must be used after insertion of RBS primer. Use Georgia Tech iGEM RBS primers (BBa_K1539021 or BBa_K1539034) or an RBS primer with a similar design.58 bp
BBa_K1539089DeviceThis primer is used to insert a low efficiency promoter to translate E. coli. genes. Must be used after insertion of RBS primer. Use Georgia Tech iGEM RBS primers (BBa_K1539021 or BBa_K1539034) or an RBS primer with a similar design.58 bp

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