beta-glucosidase gene bglX (chu_2268) from Cytophaga hutchinsonii
This is a beta-glucosidase gene (bglX, locus chu_2268) from the genome sequence of the cellulose-degrading Gram negative bacterium Cytophaga hutchinsonii.
Genome sequence of the cellulose-degrading Gram negative bacterium Cytophaga hutchinsonii.
This part codes for the enzyme Lignin Peroxidase which initiates the breakdown of lignin, a complex biopolymer in biomass. The part is currently in testing, but should produce the active enzyme. It can be used in bioenergy projects to begin the process of making biofuels. Also, there has been some discussion about the possibility of using peroxidases to break down synthetic polymers.
This part is one of a family of highly homologous genes in P.C. It was nearly impossible to isolate the gene using primers specific for the coding sequence. It was necessary to use a sequential set of PCR primers specific for the upstream and downstream regions and then the specific coding sequence, respectively.
cDNA of Phanerochaete Chrysosporium grown on Aspen Wood Culture at University of Wisconsin/USDA Forest Products Lab.
cex coding sequence encoding Cellulomonas fimi exoglucanase
The fungus ''Cellulomonas fimi'' uses an exoglucanase (from ''cex'') along with 3 endoglucanases in the degradation of cellulose into cellobiose, before use B-glucosidase to catalyse the conversion of cellobiose to D-glucose.
The original sequence had a PstI site for 781-786. We added the silent mutation CAG->CAA (Gln) by MABEL to remove this site.
Purified ''Cellulomonas fimi'' genomic DNA.
Enoly-coa hydratase from C. acetobutylicum. The sequence has been selected because it is part of an operon that has successfully been cloned into E. coli. It catalyzes the second step in butanol biosythesis. It has a His-tag added to its C terminus.
The sequence has had its codons optimized for E. coli and has had its restriction sites removed. The sequence has had its stop codon changed to TAATAA. It has had a His tag added to its c terminus.
Kwabena O. Duedu, Chris French, Sahreena Lakhundi
Exo- and endo- glucanases from the cellulolytic bacterium Cellulomonas fimi under the E. coli lac promoter. Together with betaglucosidases, the bacterium degrades cellulose into D-glucose.
Cex is a GC rich part and may require extended denaturation in amplifying the gene.
Butanol dehydrogenase from C. acetobutylicum. This catalyzes the fifth step of butanol biosythesis. It has been cloned into e. coli previously and activity has been recovered.
This part has been optimized for synthesis in E. Coli and has had its restriction sites removed. It has had its GTG start codon mutated to ATG and its stop codon mutated to TAATAA. It has also had ad C terminal His tag added.
Exo- and endo- glucanases from the cellulolytic bacterium Cellulomonas fimi under the E. coli spac promoter (BBa_J15506). Together with betaglucosidases, the bacterium degrades cellulose into D-glucose.
Cex is a GC rich part and requires extended denaturation with glycerol or commercial GC enhancers
cenA coding sequence encoding Cellulomonas fimi endoglucanase A
The cellulolytic bacterium ''Cellulomonas fimi'' uses 3 endoglucanase (including CenA) and an exoglucanase in the degradation of cellulose into cellobiose, before use B-glucosidase to catalyse the conversion of cellobiose to D-glucose.
Oxygen-tolerant hydrogenase (with RBS and terminator)
Emma Marriott, Jessica Lam, Tatum Banayat
Hydrogenase is an enzyme that catalyzes the reversible oxidation of H2. This part encodes a hydrogenase actuator taken from the large subunit of the marine bacterium Hydrogenovibrius Marinus, which has been shown to be relatively tolerant to oxygen (Kim et. Al, 2011). This part consists of a strong bicistronic design RBS (found under BioFAB ID: pFAB1655), a codon-optimized HmMBH actuator gene taken from the sequence of the large subunit of hydrogenase found in hydrogenovibrius marinus (original sequence found under F2Z6J6 at uniprot.org), and a high efficiency transcriptional terminator (found under BBa_M36281 in the Registry of Standard Biological Parts). The mechanism of the part is relatively straightforward: Once induced with the PoPS signal, the actuator will initiate protein translation and express the hydrogenase enzyme. Our actuator was used in conjunction with a rhamnose sensor (e.g. BBa_M36284).
Considerations of the benefits and convenience of having a bicsistronic design led us to choose this option, in spite of the fact that the bicistronic design is targeted for E. Coli and has not yet been tested in S. Elongatus. Interestingly enough, although the part was originally designed for expression of the hydrogenase protein in cyanobacteria, it has so far only been proved for the expression of hydrogenase protein in E. Coli. In regards to the HmMBH actuator gene, the sequence was optimized to reduce repeats and the number of sites available to restriction enzymes.
Bicistronic RBS: Mutalik VK, Guimaraes JC, Cambray G, Mai QA, Christoffersen MJ, Martin L, Yua A, Lam C, Rodriguez C, Bennett G, et al. Quantitative estimation of activity and quality for collections of functional genetic elements. Nat. Methods. 2013;10:347-353. BioFAB. (http://io.biofab.org:5000/simple/randomized_bds) Sequence for large subunit Hydrogenase: Nishihara H, Miyata Y, Miyashita Y, Bernhard M, Pohlmann A, et al. Analysis of the molecular species of hydrogenase in the cells of an obligately chemolithoautotrophic, marine hydrogen-oxidizing bacterium, Hydrogenovibrio marinus. Biosci Biotechnol Biochem. 2001;65:2780???2784. UnitPRO (http://www.uniprot.org/uniprot/F2Z6J6). High efficiency transcriptional terminator: Sunil Bodapati, Julia Joung, Daniel Esquivel-Reynoso. Part: BBa_M36281: Hard Information. Registry of Standard Biological Parts. (http://partsregistry.org/cgi/partsdb/part_info.cgi?part_name=BBa%20M36281)
B-hydroxybutyryl coa dehydrogenase from C. acetobutylicum. Catalyzes the first step in the conversion of aceto-acetylcoa into butanol. It is from an operon containing this gene as well as two other genes in the butanol production pathway. It has been successfully cloned into e. coli before and activity has been recovered. The sequence has C-terminal His-tag.
The sequence has been optimized for E. Coli and has had its restriction sites removed. A His tag has been added to the C terminus. The stop codon has been changed to TAATAA.
Genomic sequence of Chlostridium acetobutylicum. CAC_2708.
This is the butyraldehyde dehydrogenase from C. acetobutylicum. It catalyzes the fourth step in the reaction of butanol biosythesis. Its is from the pSOL plasmid of C. acetobutylicum. It also has some alcohol dehydrogenase activity.
The sequence has been optimized for use in E. Coli and has had its restriction sites removed. It has had a c-terminal His tag added and its stop codon has been changed to TAATAA.
pSOL plamid of C. acetobutylicum. CAP_0035
Butyryl coa dehyrogenase from C. acetobutylicum. The sequence has been selected because it is part of an operon that has successfully been cloned into E. coli. It catalyzes the third step in butanol biosythesis. It has a His-tag added to its C terminus.
This is the coding sequence for the heat shock protein hsp15 from E. coli
This coding sequence produces a protein that is involved in the heat shock response of E. coli. We are using this part to regulate the activity of a heat shock promoter as part of a bacterial lava lamp.
We chose this heat shock protein because it operates well in multiple bacteria including E. coli and B. subtilis and our project involves both organisms.
hsp15 is derived from the genome of E. coli K12. We retrieved the [http://BioCyc.org/ECOLI/NEW-IMAGE?type=GENE&object=G7743 sequence] from the Ecocyc database on June 26th 2009.
Endoglucanase A (CenA) from the cellulolytic bacterium Cellulomonas fimi (accession M15823) used in the degradation of cellulose into cellobiose, before using beta-glucosidase to catalyse the conversion of cellobiose to D-glucose.
RBS + Exoglucanase (cex, BBa_K118022) from the cellulolytic bacterium Cellulomonas fimi (accession M15824).
GC rich part. Extended denaturation stage in PCR with glycerol or commercial GC rich enhancers may be required
Cellulomonas fimi exoglucanase cex (accession M15824) with RBS
Carolina Guadalupe Cruz N????ez, Alejandro Alarc??n del Carmen, Franklin Cruz Villegas