Difference between revisions of "Part:BBa K2139002"

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	<partinfo>BBa_K2139002 short</partinfo>		<partinfo>BBa_K2139002 short</partinfo>
−	BBa_K2139002 encompass the coding region for an 1,4-beta-glucosidase known as Gluc1C. Gluc1C is a monomeric enzyme capable of catalyzing the removal of decomposition from cellobiose down to glucose. It can be used in conjunction with an exo-glucanase and endo-beta-1,4-glucanase for the 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 synthesized for use in Caulobacter crescentus and is codon optimized to contain a high GC content.
−	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.	+
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		+	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 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.
	<center>		<center>
	[[Image:British_Columbia_western.png|300px]]</center>		[[Image:British_Columbia_western.png|300px]]</center>
	<p>		<p>
−	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. </p>	+	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>		<p>
−	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.	+	150 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.
	<center>		<center>
	[[Image:British_Columbia_cellulase_assay.png|300px]]</center>		[[Image:British_Columbia_cellulase_assay.png|300px]]</center>
−	<p>
	Figure 2. Assay for cellulase activity with DNPC substrate.		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).</p>	+	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>
	<center>		<center>
	[[Image:British_Columbia_Cellulase.png|300px]]</center>		[[Image:British_Columbia_Cellulase.png|300px]]</center>
−	<p>Figure 3. Cellulase activity and growth assay results for C. crescentus displaying cellulases compared to C. crescentus expressing wildtype RsaA (P4A723).</p>	+	<p>Figure 3. Cellulase activity and growth assay results for <i>C.crescentus</i> displaying cellulases compared to <i>C.crescentus</i> expressing wildtype RsaA (P4A723).</p>
	<center>[[Image:British_Columbia_consortium.png|300px]]</center>		<center>[[Image:British_Columbia_consortium.png|300px]]</center>
−	<p>Figure 4:<i>C.crescentus</i> 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 <i>E.coli</i> growth is likely due to diffusion limitations and a mechanism for E.coli</p>	+	<p>Figure 4:<i>C.crescentus</i> 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 <i>E.coli</i> growth is likely due to diffusion limitations and a mechanism for <i>E.coli</i></p>
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Revision as of 20:29, 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 cultures were grown, surface proteins were extracted using low pH extraction protocol and western blot analysis were performed on extracted proteins 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 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.

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).

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