Difference between revisions of "Part:BBa K2139001"

Revision as of 20:43, 30 October 2016

Coding Sequence of Endo5a

BBa_K2139001 encompass the coding region for an endo-beta-1,4-glucanase known as Endo5A. Endo5a catalyzes the conversion of cellulose into smaller fragments by making internal cuts in the cellulose chain. It can be used in conjunction with exo-beta-1,4-glucanases for the direct conversion of cellulose into monomeric glucose. The is a monomeric protein with an optimal temperature of 50 degrees Celsius and an optimal pH ranging from 6 to 8. The genbank ascension number for this protein is HQ657203.1 (DNA), AEB00655.1 (amino acid), and a PDB code of 1VRX (homolog). This construct was synthesized by IDT for use in Caulobacter crescentus and is codon optimized for the expression in this species.

To confirm expression of surface layer fusion protein, C. crescentus cultures were grown and western blot analysis were performed on surface proteins from low pH extraction and on the cell lysates. Coomassie Brilliant Blue staining and western immunoblotting was performed. Western blots were probed with primary rabbit anti-RsaA polyclonal antibodies at 1/30,000 dilution. Goat anti-rabbit IgG was used as secondary antibody at 1/50,000 dilution. Fluorophore was detected by Odyssey Infrared Imaging System.

Figure 1. (top) Western Blot of C. crescentus cellulase expressing strains, ran on SDS-PAGE and blotted with anti-RsaA. Left to right: (1) Thermofisher ladder, (2) Gluc1C low pH extracted proteins, (3) Endo5A low pH extracted proteins, (4) E1_399 low pH extracted proteins, (5) E1_422 low pH extracted proteins, (6) ΔGCSS (ΔrsaA) low pH extracted proteins, (7) P4A723 (wildtype) low pH extracted proteins.

150 uL of each two day old culture culture was aliquoted into a clear 96 well plate(Corning) and 150 μL of assay mix (0.1 mg/ml DNPC in 50 mM pH 5.5 potassium acetate buffer) was added to the each well. OD 400 nm and OD 600 nm was measured every 30 minutes for several hours by a VarioScan plate reader (Thermo). Between measurements the culture was incubating at 30°C. Data was normalized by dividing the OD400nm measurement by the OD600nm measurement. The enzyme activity is confirmed by the increased absorbance compared to the wild type control.

Figure 2. Assay for cellulase activity with DNPC substrate.

Triplicate 5mL PYE-CM starter cultures of P4A723 (ΔrsaA C. crescentus complemented with wildtype ΔrsaA in p4A723), E1_399, E1_422, Gluc1C and Endo5A were grown in 10 mL tubes on a rotary shaker at 30°C for 2 days. Cultures were taken out of incubator and OD600 was measured. All cultures were then normalized to the lowest OD600 by diluting the remaining cultures with PYE. 100-fold dilution was used to inoculate the cultures in 200 μL well clear plate containing M2 with 0.2% w/v carboxymethylcellulose (CMC). The data indicates that the cellulase enzymes expressed, Endo5A, was able to hydrolyse cellubiose

Figure 3. Cellulase activity and growth assay results for C. crescentus displaying cellulases compared to C. crescentus expressing wildtype RsaA (P4A723).

Figure 4. Consortium growth on cellulose as the only substrate

C.crescentus expressing Endo5A was able to support it's own growth with cellulose as the sole substrate in the system. In addition, these results imply C.crescentus expressing Endo 5A cellulases were able to hydrolyze cellulose into glucose for the growth of E.coli and secondary metabolite product formation in E.coli. The wildtype control had no growth on the same substrate. This shows that the tools of our system, the Endo5A cellulase, was successful in it's purpose.

Literature data for this part can be found http://www.sciencedirect.com/science/article/pii/S1046592812003154

Sequence and Features