Part:BBa_K1321297
sfGFP fused to CBDcex driven by LacI in pSEVA331-Bb
This is a LacI-promoter expression construct of super-folder GFP fused N-terminally to CBDcex (a cellulose-binding domain; part BBa_K1321357) expressed in the broad host-range plasmid pSEVA331-Bb (part BBa_K1321300) capable of replication in G. xylinus. BBa_K1321297 is a member of the G.xylinus genetic engineering toolbox (parts BBa_K1321295 - BBa_K1321332). sfGFP expression from BBa_K1321297 has been confirmed in E.coli, as it results in low level of fluorescence 3 days after transformation (grown at 37degC standing, on LB-chloramphenicol plates) even without the addition of lactose. BBa_k1321297 is currently being characterized in G.xylinus.
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). As no registry parts had been tested in G.xylinus, the aim of this toolbox was to determine the parts usable in G.xylinus and to characterize them. 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 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.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal prefix found in sequence at 4159
Illegal suffix found in sequence at 1288 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 4159
Illegal SpeI site found at 1289
Illegal PstI site found at 1303
Illegal NotI site found at 1296
Illegal NotI site found at 4165 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 4159
- 23INCOMPATIBLE WITH RFC[23]Illegal prefix found in sequence at 4159
Illegal suffix found in sequence at 1289 - 25INCOMPATIBLE WITH RFC[25]Illegal prefix found in sequence at 4159
Illegal XbaI site found at 4174
Illegal SpeI site found at 1289
Illegal PstI site found at 1303
Illegal NgoMIV site found at 230
Illegal NgoMIV site found at 2478 - 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 245
| None |