Linker-cipA-linker + phytochelatin (PC) EC20 with LacI promoter
Linker-CBDcipA-Linker + Phytochelatin (PC) EC20
Please see part pages for the composite parts
Synthetic phytochelatin EC20 (metal binding peptide) fused C-terminally to CBDcipA (a cellulose-binding domain), which contains an endogenous N and C-terminal linker sequence. At present the site-directed mutagenesis for this construct is in progress to correct an illegal EcoRI site which was identified in the CBDcipA. This construct is part of a library of fusions with cellulose binding domains which we designed to bind to cellulose and enable capture of heavy metals ([http://2014.igem.org/Team:Imperial/Functionalisation project page]). Other fusion parts with this metal binding protein can be seen in the table below: Note that the start and stop codon, plus 6 bp either side of the sequence, are included the RFC25 prefix and suffix which is not shown.
The linker sequences mean fusions should be fine either N or N-terminally to the CBDcipA
This sequence is produced by an assembly method.
This sequence is produced by RFC10-assembly of a RFC10-RBS with a RFC25-prefixed part.
The resulting sequence is a combination of the RFC10 RBS-CDS scar (tactag) and 9 nucleotides from the RFC25 prefix (atg gcc ggc).
RBS (Elowitz 1999) -- defines RBS efficiency
Varies from -6 to +1 region from original sequence to accomodate BioBricks suffix. No secondary structures are formed in the given RBS region. Users should check for secondary structures induced in the RBS by upstream and downstream elements in the +50 to -50 region, as such structures will greatly affect the strength of the RBS. Contact info for this part: Brian Chow
Vinay S Mahajan, Voichita D. Marinescu, Brian Chow, Alexander D Wissner-Gross and Peter Carr IAP, 2003.
The Plac insert was PCR'd from the MG1655 strain of E.coli K12.
Inverting regulatory region controlled by LacI (<bb_part>BBa_C0010</bb_part>, <bb_part>BBa_C0011</bb_part>, etc.) The pLac regulatory region is a 243 base-pair sequence with standard BioBrick prefix and suffix sections on its ends. It contains two protein binding sites: CAP, which is generally present in E.coli and is assocciated with cell health and availability of glucose., and LacI, the Lac inhibitor <bb_part>BBa_C0010</bb_part> which binds in an dimerized cooperative manner to inhibit the transcription of the protein that follows. In the presence of lactose or IPTG, an analog of lactose, LacI is unable to correctly bind and inhibit transcription. This allows <bb_part>BBa_R0010</bb_part> to be used as a inverter or as a detector of lactose or IPTG.
LacI binds to this regulator. This part is incompatible with species containing active LacI coding regions. Lactose and IPTG disable the operation of LacI and this regulator. This part is incompatible with environments containing lactose or lactose analogs.
This is the scar produced by assembly using RFC 25. If you are assembling a composite part using RFC 25, you can insert this part and specify blunt assembly to get the desired sequence.
Sequence is not natural. Basis for the sequence was from the following reference: Bae W, Chen W, Mulchandani A, Mehra RK (2000) Enhanced bioaccumulation of heavy metals by bacterial cells displaying synthetic phytochelatins. Biotechnol Bioeng. 70(5):518-24.
Synthetic Phytochelatin EC(20) rfc25 fusion format
The designed construct was ordered from as a GeneArt?? String (Invitrogen??? Life Technologies).
This CBD is from the Cellulosomal -scaffolding protein A (cipA) of Clostridium thermocellum including the endogenous linker sequences at the N and C-terminus (UniProt ID Q06851 link http://www.uniprot.org/uniprot/Q06851). It is in RFC25 format to allow for easy use in protein fusions. The CBD is part of the CBM3 family (link to cazy http://www.cazy.org/CBM3.html). This CBD has been used in many application: in fusions with cell adhesion peptides to enhance the properties of cellulose as a cell-growth matrix (Andrade et al 2010a, Andrade et al 2010b), fused to enzymes to remove contaminants from water (Kauffmann et al 2000) and fused to an antimicrobial peptide (Ramos, Domingues & Gama 2010). At present the cloning for these constructs is still in progress to correct an illegal EcoRI site which was identified in the parts with this CBD. This can be achieved with a silent mutation via site-directed mutagenesis and we aim to send these parts to the registry once this is complete.
The DNA sequence from the relevant portion of http://www.ncbi.nlm.nih.gov/nuccore/144777 was codon optimised for ''E.coli''. RFC[25] prefix and suffix were appended to the sequence with an additional 4 basepairs (gatc) at the beginning and end to allow space for the restriction enzymes to bind to the EcoRI and PstI sites at the ends of the sequence for cloning purposes.
Conversion of the iGEM parts registry to SBOL2.1