Difference between revisions of "Part:BBa K2139002"

Latest revision as of 20:45, 30 October 2016

Coding Sequence of Gluc1C

BBa_K2139002 encompass the coding region for an 1,4-beta-glucosidase known as Gluc1C from Paenibacillus sp. MTCC 5639 bacteria. Gluc1C is a monomeric enzyme capable of breaking cellobiose down to glucose. It can be used in conjunction with an exo-glucanase and endo-beta-1,4-glucanase for the complete conversion of cellulose to glucose. The genbank ascension number for this protein is JQ713769.1 (DNA), AFQ36783.1 (amino acid), and a PDB code of 2O9R_A. This construct was codon optimized for the expression in C.crescentus and it was synthesized by IDT. To confirm expression of surface layer fusion protein, C.crescentus culture expressing Gluc1C was grown, surface proteins were extracted using low pH extraction protocol and western blot analysis were performed on extracted proteins. 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). The expression of Gluc1C-rsaA fusion on cell surface was confirmed.

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.

To test activity of Gluc1C, the test with DNP-cellobiose substrate was performed. 150 μL of Gluc1C and rsaA C.crescentus cultures were 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 cultures were incubating at 30°C. Data was normalized by dividing the OD400nm measurement by the OD600nm measurement. Gluc1C showed cellulotytic activity compare to the rsaA control(Figure 2).

Figure 2. Assay for cellulase activity with DNPC substrate.

To further validate expression of cellulases on surface layer triplicate 5mL PYE-CM starter cultures C.crescentus expressing Gluc1C, E1_399, E1_422, Eno5A and rsaA 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 growth of cultures was monitored over 4 days. The co-culture of endoglucanases with Gluc1C showed significant growth on CMC.

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

Figure 4:C.crescentus expressing Gluc1C was able to support it's own growth with cellulose as the sole substrate in the system. The wildtype control had no growth on the same substrate. Lack of E.coli growth is likely due to diffusion limitations and a mechanism for E.coli

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 264
1000
INCOMPATIBLE WITH RFC[1000]
Illegal SapI.rc site found at 1138

@@ Line 9: / Line 9: @@
 BBa_K2139002 encompass the coding region for an 1,4-beta-glucosidase known as Gluc1C from <i>Paenibacillus sp.</i> MTCC 5639 bacteria. Gluc1C is a monomeric enzyme capable of breaking cellobiose down to glucose. It can be used in conjunction with an exo-glucanase and endo-beta-1,4-glucanase for the complete conversion of cellulose to glucose. The genbank ascension number for this protein is JQ713769.1 (DNA), AFQ36783.1 (amino acid), and a PDB code of 2O9R_A. This construct was codon optimized for the expression in <i>C.crescentus</i> and it was synthesized by IDT.
-To confirm expression of surface layer fusion protein, <i>C.crescentus</i> cultures were grown, surface proteins were extracted using low pH extraction protocol and western blot analysis were performed on extracted proteins. 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.
+To confirm expression of surface layer fusion protein, <i>C.crescentus</i> culture expressing Gluc1C was grown, surface proteins were extracted using low pH extraction protocol and western blot analysis were performed on extracted proteins. 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). The expression of Gluc1C-rsaA fusion on cell surface was confirmed.
 <center>
 [[Image:British_Columbia_western.png|300px]]</center>
@@ Line 15: / Line 15: @@
 Figure 1. (top) Western Blot of <i>C.crescentus</i> 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. </p>
 <p>
-μL of E1_399, E1_422, Gluc1C, Endo5A and rsaA cultures as a control 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. Gluc1C showed cellulotytic activity compare to the rsaA control.
+To test activity of Gluc1C, the test with DNP-cellobiose substrate was performed. 150 μL of Gluc1C and rsaA  <i>C.crescentus</i> cultures were 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 cultures were incubating at 30°C. Data was normalized by dividing the OD400nm measurement by the OD600nm measurement. Gluc1C showed cellulotytic activity compare to the rsaA control(Figure 2).
 <center>
 [[Image:British_Columbia_cellulase_assay.png|300px]]</center>
 Figure 2. Assay for cellulase activity with DNPC substrate.
-Triplicate 5mL PYE-CM starter cultures of P4A723 (ΔrsaA  <i>C.crescentus</i> 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).</p>
+To further validate expression of cellulases on surface layer triplicate 5mL PYE-CM starter cultures <i>C.crescentus</i> expressing Gluc1C, E1_399, E1_422, Eno5A and rsaA 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 growth of cultures was monitored over 4 days. The co-culture of endoglucanases with Gluc1C showed significant growth on CMC. </p>