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

Abstract

Lignocellulosic biomass,the most abundant renewable resources in nature, represents a promising alternative to fossil fuels for sustainable production of chemicals and material. However, the main technological obstacle for industrial exploitation of biomass is the recalcitrant nature of lignocellulosic polymers. One approach to harness the energy contained in biomass is to convert the lignocellulosic polymers in simple sugars, which can be further transformed in valuable compounds, using microorganisms. Some anaerobic microorganisms have developed approaches to break down recalcitrant plant biomass using elaborate extracellular enzyme complexes, containing a scaffolding protein with many attached cellulolytic enzymes. The assembly of such complexes requires engineering of highly specific cohesin-dockerin interactions. Our approach focuses on adapting the surface layer of Caulobacter crescentus for the for highly efficient display of cellulolytic enzymes. We specifically chose C. crescentus as it natively expresses a two dimensional crystal lattice protein called a surface layer (S-Layer), which can be engineered to work as an enzyme display. Our goals were to embed cellulolytic enzymes into the S-Layer protein to confirm their proper folding and activity. We believe that our approach will simplify the process of "cellulosome" engineering by exploiting the robust secretion and display system of Caulobacter. The developed platform can be easily adapted for the display of different enzymatic activities.

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