Difference between revisions of "Team:British Columbia/Project/S-Layer"

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.

For the cloning of CEX: P4A723 rsaA plasmid and was digested using Bgl II and Nhe I restriction enzymes while CEX was digested using Bll II and Nhe I 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.

Transformed colonies were then plated on and selected from a LB-CM plate and grown overnight in LB-CM media. Recombinant plasmid was then removed using ,a href="#">QIAGEN miniprep kit and sent for sequence confirmation.

After sequence confirmation, the mini preped plasmids were electroporated into electro compitent C. Crescentus and colonies were grown on a PYE-CM plate. One colony was selected and streaked onto a fresh plate, which would be used for all future assays.

Cloning of cellulase enzymes into psb1c3 with ptac promoter and rbs

Synthesized cellulase enzymes were amplified using high fidelity Low pH extraction (Walker et al. 1992) was performed using different pHs of HEPES buffer to remove only crystalline s-layer without lysing cells. Proteins were then stored in epindorf tubes at -20°C to be used at a later date.

Surface layer fusion protein expression confirmation

Caulobacter Cellulase Activity Analysis

For cellulase enzyme activity measurement, triplicate 5mL PYE-CM starter cultures were grown at 30°C in 10 mL tubes shaking for 2 days. Cultures were taken out of incubator and optical density at 600 nm was measured. All cultures were then normalized to the lowest OD 600 nm by diluting the remaining culture with PYE. 1 mL of each sample was removed and washed 3 times with fresh PYE to remove and lysed cells that may interfere with cellulase activity results.

Two separate conditions were tested using the Cellulase Activity Assay Protocol adopted from (): one with washed cells and the other with unwashed cells (all samples had been normalized previously). 150 uL of culture from the two separate conditions were aliquoted into a clear 96 well plate then 150 uL assay mix (0.1 mg/ml DNPC in xx M pH 5.5 potassium acetate buffer) was added into the each occupied well.

OD 400 nm was measured every 30 minutes for 5 hours by an XX plate reader. Between measurements the culture was incubating at 30°C.

Results

References