"
Team:WPI-Worcester/Our-Construct
From 2014.igem.org
| (One intermediate revision not shown) | |||
| Line 626: | Line 626: | ||
</br> | </br> | ||
<h4>Our Construct</h4><p><center><img src="https://static.igem.org/mediawiki/2014/thumb/e/e0/WPI_OurConstruct.png/800px-WPI_OurConstruct.png"/></center></p></br><p>The construct developed in this project is a combination of the N-terminal domain of BclA and the CAEV p28 antigen. Both sequences were ordered as codon-optimized oligos, so that <i>B. anthracis</i> and CAEV would not have to be worked with directly. The sequence encoding the N-terminal domain of BclA is ligated to the sequence for the CAEV p28 antigen. This plasmid is then combined with a ribosome binding site, a constitutive promoter, and a double terminator in order to make a complete construct. The resulting plasmid allows the <i>E. coli</i> it is transformed into to express the CAEV p28 antigen connected to the N-terminal domain of BclA, which is anchored in the outer surface of the plasma membrane. This plasmid is transformed into competent <i>E. coli</i>, which then express CAEV on their membrane surface, thanks to the anchoring of BclA’s N-terminal domain. </p> | <h4>Our Construct</h4><p><center><img src="https://static.igem.org/mediawiki/2014/thumb/e/e0/WPI_OurConstruct.png/800px-WPI_OurConstruct.png"/></center></p></br><p>The construct developed in this project is a combination of the N-terminal domain of BclA and the CAEV p28 antigen. Both sequences were ordered as codon-optimized oligos, so that <i>B. anthracis</i> and CAEV would not have to be worked with directly. The sequence encoding the N-terminal domain of BclA is ligated to the sequence for the CAEV p28 antigen. This plasmid is then combined with a ribosome binding site, a constitutive promoter, and a double terminator in order to make a complete construct. The resulting plasmid allows the <i>E. coli</i> it is transformed into to express the CAEV p28 antigen connected to the N-terminal domain of BclA, which is anchored in the outer surface of the plasma membrane. This plasmid is transformed into competent <i>E. coli</i>, which then express CAEV on their membrane surface, thanks to the anchoring of BclA’s N-terminal domain. </p> | ||
| + | <p>Using the BclA construct, we also expressed YFP on the surface of <i>E.coli</i>, as can be seen in the image below.The <i>E.coli</i> with YFP extracellulary expressed was used in our proof of principle experiments. This is because it is easier to monitor YFP using microscopy and the YFP also had a known antibody we could test our agglutination assay with. The results of this can be found at <a href="https://2014.igem.org/Team:WPI-Worcester/Proof-of-Principle"> this link.</a></p></br> | ||
| + | <p> | ||
<p><center><img src="https://static.igem.org/mediawiki/2014/8/81/WPI_YFPconstruct.png"/></center></p> | <p><center><img src="https://static.igem.org/mediawiki/2014/8/81/WPI_YFPconstruct.png"/></center></p> | ||
| - | + | <h9>Design Considerations</h9></p> | |
| - | + | ||
<p>When designing this construct we had a few important considerations. First we worked with proteins from two disease related organisms. The first is the BclA protein from <i>Bacillus anthracis</i>. This protein normally contains three domains, we only took the first of these domains in order to minimize the DNA we were taking from this pathogenic organism. The second protein we used was from the Caprine Arthritis Encephalitis Virus (CAEV). We used the p.28 domain of a hairlike capsid protein from CAEV. Again, we did not use the entire protein in order to minimize the possible virulence.</p> | <p>When designing this construct we had a few important considerations. First we worked with proteins from two disease related organisms. The first is the BclA protein from <i>Bacillus anthracis</i>. This protein normally contains three domains, we only took the first of these domains in order to minimize the DNA we were taking from this pathogenic organism. The second protein we used was from the Caprine Arthritis Encephalitis Virus (CAEV). We used the p.28 domain of a hairlike capsid protein from CAEV. Again, we did not use the entire protein in order to minimize the possible virulence.</p> | ||
<p>Furthermore, we had to make sure all of our domains would remain in frame once the constructs were assembled. This meant taken scar site length into consideration when order the oligos which we used to create our new biobricks. Our construct would be useless if the antigen was no longer in frame once it was attached to the N-terminal domain of BclA.</p> | <p>Furthermore, we had to make sure all of our domains would remain in frame once the constructs were assembled. This meant taken scar site length into consideration when order the oligos which we used to create our new biobricks. Our construct would be useless if the antigen was no longer in frame once it was attached to the N-terminal domain of BclA.</p> | ||
Latest revision as of 23:19, 17 October 2014
"
Team:WPI-Worcester
From 2014.igem.org
Our Construct
The construct developed in this project is a combination of the N-terminal domain of BclA and the CAEV p28 antigen. Both sequences were ordered as codon-optimized oligos, so that B. anthracis and CAEV would not have to be worked with directly. The sequence encoding the N-terminal domain of BclA is ligated to the sequence for the CAEV p28 antigen. This plasmid is then combined with a ribosome binding site, a constitutive promoter, and a double terminator in order to make a complete construct. The resulting plasmid allows the E. coli it is transformed into to express the CAEV p28 antigen connected to the N-terminal domain of BclA, which is anchored in the outer surface of the plasma membrane. This plasmid is transformed into competent E. coli, which then express CAEV on their membrane surface, thanks to the anchoring of BclA’s N-terminal domain.
Using the BclA construct, we also expressed YFP on the surface of E.coli, as can be seen in the image below.The E.coli with YFP extracellulary expressed was used in our proof of principle experiments. This is because it is easier to monitor YFP using microscopy and the YFP also had a known antibody we could test our agglutination assay with. The results of this can be found at this link.
When designing this construct we had a few important considerations. First we worked with proteins from two disease related organisms. The first is the BclA protein from Bacillus anthracis. This protein normally contains three domains, we only took the first of these domains in order to minimize the DNA we were taking from this pathogenic organism. The second protein we used was from the Caprine Arthritis Encephalitis Virus (CAEV). We used the p.28 domain of a hairlike capsid protein from CAEV. Again, we did not use the entire protein in order to minimize the possible virulence.
Furthermore, we had to make sure all of our domains would remain in frame once the constructs were assembled. This meant taken scar site length into consideration when order the oligos which we used to create our new biobricks. Our construct would be useless if the antigen was no longer in frame once it was attached to the N-terminal domain of BclA.
