Part:BBa_K1321295
CBDclos fused to sfGFP driven by LacI in pSEVA331-Bb
A lacI-promoter expression construct of super-folder GFP fused C-terminally to CBDclos (part BBa_K1321360) in pSEVA331-Bb backbone (part BBa_K1321300). Expression of sfGFP-CBDclos from BBa_k1321295 has been confirmed in E.coli (it results in bright fluorescence after 2 days of incubation on LB-chloramphenicol plates) and is currently being characterized in G.xylinus.
This construct is part of a library of super-folder GFP fusions with cellulose binding domains, which we used to assay the CBD binding affinity and is a member of the G.xylinus genetic engineering toolkit (parts BBa_K1321295 - BBa_K1321332).
G.xylinus toolkit was designed to ease genetic engineering of cellulose-based biomaterials using the cellulose synthesizing bacterium Gluconacetobacter xylinus (parts Bba_K1321305 and BBa_K1321306). As no registry parts had been tested in G.xylinus, the aim of this toolkit was to determine the parts usable in G.xylinus and to characterize them in this host. 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 because 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 toolkit 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 toolkit available for the synthetic biology community, Imperial iGEM 2014 team has made it freely available upon request, with quality control provided (see Experience). To request, please contact Imperial iGEM 2014 team.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal prefix found in sequence at 4132
Illegal suffix found in sequence at 1261 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 4132
Illegal SpeI site found at 1262
Illegal PstI site found at 1276
Illegal NotI site found at 1269
Illegal NotI site found at 4138 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 4132
- 23INCOMPATIBLE WITH RFC[23]Illegal prefix found in sequence at 4132
Illegal suffix found in sequence at 1262 - 25INCOMPATIBLE WITH RFC[25]Illegal prefix found in sequence at 4132
Illegal XbaI site found at 4147
Illegal SpeI site found at 1262
Illegal PstI site found at 1276
Illegal NgoMIV site found at 230
Illegal NgoMIV site found at 2451 - 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 551
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