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.
Kwabena O. Duedu, Chris French, Sahreena Lakhundi
The DNA was amplified from E. coli BL21 genomic DNA using primers based on published sequence (Genbank accession J01636, gi:146575). The annotation shown here is based on that associated with this Genbank entry. The sequence shown here is derived by sequencing the construct.
This part (submitted in pSB1A2) consists of the lac promoter and lacZ' gene, encoding the N-terminal 76 amino acid residues of LacZ, sufficient to complement the lacZ-delta-M15 mutation for blue-white selection on Xgal plates. A SacI site has been introduced at the 3' end, overlapping the XbaI site of the Biobrick prefix. This is designed to be used as a cloning vector for making new biobricks. PCR primers can be designed with a SacI site in one primer and an SpeI site in the other. This removes the necessity for an excessively long non-complementary tail on one primer bearing either the full biobrick prefix or suffix. The PCR product can then be digested with SacI and SpeI for insertion into this plasmid, replacing the Plac-lacZ' cassette. Recombinant plasmids will then be white on IPTG/Xgal plates, whereas any that still contain the original insert will be blue. We have used this strategy to prepare several biobricks, including BBa_J33204, which contains the xylE gene encoding catechol-2,3-dioxygenase. (For making biobricks that contain lacZ', BBa_J33204 can be used in the same way; in this case, clones with plasmids that still contain xylE will turn yellow on addition of a drop of 10 mM catechol.)
Note that the SacI site overlaps the SpeI site. The Biobrick prefix ends ...TCTAGAG. When this is added to the CTC at the start of the sequence shown here, the SacI site, GAGCTC, is generated.
Cellulomonas fimi exoglucanase cex (accession M15824) with RBS
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
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.
strong synthetic E. coli ribosome binding site with SacI site.
This is a strong synthetic E. coli ribosome binding site. It is synthesised as two complementary oligonucleotides rather than being incorporated into a biobrick plasmid. It incorporates a SacI site overlapping the XbaI site, which is preserved when it is added to any other biobrick. This allows easy detection of the RBS after it has been added upstream of a biobrick coding sequence in a plasmid vector.
Note the presence of a SacI site overlapping the XbaI site, which is preserved when this biobrick is added to any other biobrick. At the time of writing, this biobrick is added as a short piece of DNA composed of two complementary oligonucleotides rather than being incorporated into a biobrick cloning vector by itself. It can be added upstream of a biobrick coding sequence, and its presence can easily be detected in miniprep DNA on a gel by using a SacI-SpeI or similar digest.
cex coding sequence encoding Cellulomonas fimi exoglucanase
Purified ''Cellulomonas fimi'' genomic DNA.
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.
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.
Conversion of the iGEM parts registry to SBOL2.1