sfGFP fused to CBDclos driven by LacI in pSEVA331-Bb
A LacI-promoter expression construct of super-folder GFP fused N-terminally to CBDclos- a cellulose binding domain(part BBa_K1321356) in broad host-range plasmid pSEVA331-bb (part BBa_K1321300) which replicates in the cellulose producing bacterium G.xylinus BBa_K1321356 is part of a library of Super-folder GFP fusions with cellulose binding domains, which we used to assay the CBD binding affinity. BBa_K1321356 in pSEVA331-Bb is part of the G.xylinus genetic engineering toolbox. G.xylinus toolbox was designed to ease genetic engineering of cellulose-based biomaterials using the cellulose synthesizing bacterium Gluconacetobacter xylinus (parts Bba_K1321305 and BBa_K1321306) by providing a collection of widely used parts in pSEVA331-Bb backbone. pSEVA331-Bb is a non-standard broad host range plasmid capable of replication in G.xylinus and E.coli (a shuttle vector) and was selected as the registry's standard plasmid backbone pSB1C3 can not be used for G.xylinus engineering. NOTE: Because the registry's standard plasmid backbone pSB1C3 is not capable of replication in Gluconacetobacter species, the G.xylinus genetic engineering toolbox is housed mainly in pSEVA331-Bb. pSEVA331-Bb is a non-standard backbone, which therefore can't be quality controlled by and maintained in the registry. However, in order to make the G.xylinus toolbox available for the synthetic biology community, Imperial iGEM 2014 team has made the toolbox freely available upon request, with quality control provided (see Experience). For requests, please contact Imperial iGEM 2014 team.
BBa_K1321296 was created by restricting BBa_K1321356 and BBa_K1321300 with PstI and XbaI, gel purifying the resulting fragments, ligating using T4 DNA ligase and transforming the ligase products into chemically competent DH10B cells.
pSEVA331-Bb - pSEVA331 with biobrick prefix and suffix
The original pSEVA331 backbone comes from the SEVA (Standard European Vector Architecture) collection.
This is pSEVA331 converted into a biobrick compatible format by substituting the original multiple cloning site for biobrick prefix and suffix. pSEVA331 is a broad host range plasmid, capable of replication in E.coli and several other organisms. It is capable of replication in the cellulose producing bacterium Gluconacetobacter xylinum. We have used it as our standard backbone for G.xylinum genetic engineering.
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.
Re-entry of BBa_K863111, Cellulose Binding Domain CBDclos in rfc25
This is a direct copy of BBa_K863111 with slight modifications to the beginning and end of the sequence for the purposes of allowing composite parts to be added correctly using this part.
This part is a copy of BBa_K863111 (Cellulose binding domain of C. cellulovorans cellulose binding protein gene (Freiburg-Standard)) and was created only as a registry entry, removing the atg-NgoMIV parts at the front and the AgeI-taa basepairs from the sequence so that the registry does not flag it as rfc25 incompatible, and it enables us to enter fusion proteins with this part as automatic composite parts in the method as described by: http://2009.igem.org/Team:Freiburg_bioware/general.
sfGFP in Freiburg format (RFC 25) to allow for easy use in fusion proteins.
Conversion of the iGEM parts registry to SBOL2.1