Difference between revisions of "Team:British Columbia/Project/S-Layer"
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<h2 class="page-header">Methods</h2> | <h2 class="page-header">Methods</h2> | ||
<h3>Cloning of cellulase enzymes into rsaA plasmid in C. crescentus</h3> | <h3>Cloning of cellulase enzymes into rsaA plasmid in C. crescentus</h3> | ||
| − | <p>Synthesized cellulase enzymes were amplified using high fidelity <a href="#">phusion polymerase chain reaction</a>. Cellulase enzymes were amplified in | + | <p>Synthesized cellulase enzymes were amplified using high fidelity <a href="#">phusion polymerase chain reaction</a>. Cellulase enzymes were amplified in 100 ul reactions using the following primers to add the restriction enzyme cut sites (Bgl II, Pst I): |
Endo5A Primers: AAAAAAAAAAAAAAAAAAAAA | Endo5A Primers: AAAAAAAAAAAAAAAAAAAAA | ||
Gluc1C Primers: AAAAAAAAAAAAAAAAAAAAA | Gluc1C Primers: AAAAAAAAAAAAAAAAAAAAA | ||
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CEX Primeers: AAAAAAAAAAAAAAAAAAAAA (addition of Bgl II and Nhe I) | CEX Primeers: AAAAAAAAAAAAAAAAAAAAA (addition of Bgl II and Nhe I) | ||
</p> | </p> | ||
| + | <p>pSB1C3 high copy assembly plasmid was <a href="#">digested</a> using ___ and ___ restriction enzymes while Endo5A, Gluc1C, E1, G12, and CEX were <a href="#">digested</a> using ___ and ___ restiction enzymes. The plasmid digest was then purified by agarose gel purification using ____ kit while the gene digest was purified by PCR purification using ___ kit. Both purified digest were <a href="#">ligated</a> together and ligation mix was then transformed in chemically competent dh5a E. coli. <p> | ||
<h3></h3> | <h3></h3> | ||
<h2 class="page-header">Results</h2> | <h2 class="page-header">Results</h2> | ||
Revision as of 14:59, 3 October 2016
S-Layer Engineering
Abstract
Lignocellulosic biomass, providing the structural support for plants, is the earth's largest raw available reserve of carbon for bioconversion. Composed of cellulose, hemicellulose, and lignin, lignocellulosic biomass is evolved to withstand the elements and protect plants from degradation during their lifespans. These properties make it extremely difficult to degrade lignocellulosic biomass, with only a few cellulolytic organisms being able to degrade it naturally. Scientific approaches to degrade lignocellulose into valorized materials have mainly been focused on...............................this is a problem because................ Our approach focuses on displaying lytic enzymes on the surface of the bacterium Caulobacter crescentus in the hopes that it will increase the speed of cleavage and will release more glucose into the system for E. coli to convert into valorized chemicals. We specifically chose C. crescentus as it natively expresses a two dimensional crystal lattice protein called a surface layer (S-Layer). Our goals were to clone in our cellulolytic enzymes into the S-Layer protein in the hopes when the bacterium expresses the S-Layer protein our enzymes will be embedded onto the this protein and folded correctly on the surface of the bacterium.
Key Achievements
- Cloned in 5 different cellulase constructs onto the rsaA plasmid and transformed into C. crescentus: Endo5A, Gluc1C, E1_422, E1_399, CEX
- Bio Bricked 5 different cellulase enzymes into PSB1C3 to with pTac promoter and RBS: Endo5A, Gluc1C, E1, G12, CEX
- Confirmed surface layer fusion protein expression of 5 different cellulase constructs: Endo5A, Gluc1C, E1_422, E1_399, CEX
- Confirmed increased cellulase activity of five different cellulase constructs expressed on fusion protein of C. crescentus: Endo5A, Gluc1C, E1_422, E1_399, CEX
- Confirmed baseline intracellular cellulase activity of five different cellulase constructs expressed in e. Coli: Endo5A, Gluc1C, E1, Gluc1C, CEX
Design
Methods
Cloning of cellulase enzymes into rsaA plasmid in C. crescentus
Synthesized cellulase enzymes were amplified using high fidelity phusion polymerase chain reaction. Cellulase enzymes were amplified in 100 ul reactions using the following primers to add the restriction enzyme cut sites (Bgl II, Pst I): Endo5A Primers: AAAAAAAAAAAAAAAAAAAAA Gluc1C Primers: AAAAAAAAAAAAAAAAAAAAA E1_422 Primers: AAAAAAAAAAAAAAAAAAAAA E1_399 Primers: AAAAAAAAAAAAAAAAAAAAA G12 Primers: AAAAAAAAAAAAAAAAAAAAA CEX Primeers: AAAAAAAAAAAAAAAAAAAAA (addition of Bgl II and Nhe I)
pSB1C3 high copy assembly plasmid was digested using ___ and ___ restriction enzymes while Endo5A, Gluc1C, E1, G12, and CEX were digested using ___ and ___ restiction enzymes. The plasmid digest was then purified by agarose gel purification using ____ kit while the gene digest was purified by PCR purification using ___ kit. Both purified digest were ligated together and ligation mix was then transformed in chemically competent dh5a E. coli.
