These parts were biobricked by Beilefeld 2012 (link to their wiki https://2012.igem.org/Team:Bielefeld-Germany/Results/cbc), please see their part pages (link BBa_K863111 and BBa_K863101) for detailed information. CBDclos is from the cellulolytic bacterium Clostridium cellulovorans and CBDcex originates from the cellulolytic bacterium Cellulomonas fimi. This is the same organism as for CBDcenA; cex is from the exoglucanase gene and cen is from the endoglucanase gene.

This part, BBa_K1321340 (link http://parts.igem.org/Part:BBa_K1321340) is based on the double cellulose-binding domain construct synthesised and characterised by Linder et al1996, who found that this double CBD had higher affinity for cellulose than either of the two CBDs on their own. The main difference is that our part contains an additional linker sequence on the N-terminus of the protein.

The two CBDs are from the fungus Trichoderma reesei (Hypocrea jecorina) Exocellobiohydrolase (Exoglucanase) I (cbh1), uniprot ID P62694; and Exocellobiohydrolase (Exoglucanase) II, uniprot ID P07987 (cbh2); with a linker peptide between the two CBDs and at the N-terminus of the protein. Both linkers are the same amino acid sequence and are based on the endogenous linker sequences that exists in cbh1 and cbh2 genes. The linker sequence is PGANPPGTTTTSRPATTTGSSPGP which is the same as used by Linder et al 1996. The first three amino acids are from the cbh2 endogenous linker, and the rest is from the cbh1 endogenous linker. CBDcbh1 is placed C-terminal to CBDcbh2 because naturally CBDcbh1 is a C-terminal domain and CBDcbh2 is an N-terminal domain. Both CBDs are from the CBM family 1 (link to cazy here). The precise location of the CBD within the cbh genes was slightly different according to the uniprot annotations and the sequence used by Linder et al 1996; we chose to use the sequence from the paper since the protein was expressed and characterised successfully.

Description to be completed. An illegal EcoRI site was identified in these parts. A silent mutation can correct this and site-directed mutagenesis is currently in progress with the aim to submit these parts soon.

This CBD is from the Endoglucanase A (cenA) gene from Cellulomonas fimi and also contains an endogenous C-terminal linker. The cellulose binding domain region occurs at the N-terminus of the cenA gene (UniProt P07984 link to http://www.uniprot.org/uniprot/P07984) and is from the CBM family 2 (link to http://www.cazy.org/CBM2.htm)

The linker sequence (PTTSPTPTPTPTTPTPTPTPTPTPTPTVTP) is Pro-Thr box which has an extended conformation and acts as a hinge region (Gilkes et al 1989). The endoglucanase CBDcenA has 50% homology to the exoglucanase CBDcex (BBa_K863101) and the linker is highly conserved in both, but the order of the catalytic, linker and cellulose-binding regions is reversed (Warren et al 1986). It has been shown that CBDcenA has the highest binding affinity for crystalline cellulose out of the C. fimi CBDs (Kim et al 2013).

These two parts already existed in the BioBrick registry, but since we needed to use them as fusions with the CBDs we improved the parts by making them compatible with the RFC25 (link) Frieburg Fusion BioBrick standard.

Metallothioneins (MTs) are genetically encoded cysteine-rich peptides which serve a role in the detoxification of heavy metals in plants, animals and some prokaryotes (Cobbett, 2002). They have a similar structure and function to phytochelatins, but are produced by different mechanisms (Cobbett, 2002). SmtA is from the cyanobacteria Synechococcus sp. and was used by the Tokyo-NoKoGen 2011 igem team (link) (who submitted the RFC10 part originally, BBa_K519010) to confer cadmium (Cd2+) tolerance to E.coli cells when expressed. fMT originates from the seaweed Fucus vesiculosus and was submitted by Groningen team 2009 (link) and detailed information can be found on the original part page (BBa_K190019).

We successfully cloned the following CBD fusion constructs: (insert table when finalised)

This protein is the histidine-rich nickel-binding protein from Heliobacter pylori (GenBank accession ACX98466.1), and already existed as a BioBrick in the registry (link to K1151001). Since we needed to use this part in fusion proteins we improved the part by submitting it as a basic part in RFC(25) (link) Freiberg fusion format (link here to K1321009).

We successfully cloned (link to cloning protocols on the word cloned) the following CBD fusion constructs with NiBP: (insert table from the parts table spreadsheet when finalised)

However, sadly we discovered late on in the project that the DNA sequence of the NiBP protein in the registry is out of frame, leading to an incorrect protein translation and read-through of the stop codon (figure 2).

Further investigation into the error let us to identify a single nucleotide deletion in BBa_K1151001 compared to the native H.pylori DNA sequence (GenBank CP000012.1 (link http://www.ncbi.nlm.nih.gov/nuccore/261837457) bp1391851-1392033, reverse complement strand). As seen in Figure 3 the deletion is a ‘C’ at bp 53 of the native sequence. This error was propagated through all our NiBP constructs and means that any subsequent C-terminal fusions are also out of frame and we were unable to express and characterise these constructs. We have noted this on the description pages for each of the parts and hope that in the future they will still be useful, since the error could be corrected relatively easily by a team who wishes to use them by site-directed-mutagenesis PCR to insert the missing nucleotide.