Part:BBa_K4368000
pcstA + rbs + yebF + cenA + terminator
Description
CenA encodes for the endoglucanase gene of Cellulomonas fimi (BBa_K118023). This enzyme is responsible of the degradation of cellulose working coordinated with the genes cex and bglX. In addition, this part includes the composition used by the team, which includes a strong rbs (BBa_B0030), a double terminator (BBa_B0015) as well as a promoter inducible by glucose concentration (BBa_K118011). Furthermore, we improve this composite adding a gen encoding a motor protein named YebF (BBa_K1610300) that secrete the enzyme out of the E. coli membrane. The gene has been placed under the control of this promoter to build the glucose concentration-based gene regulatory circuit that integrates all our parts.
Characterization
The expression cassette sequence was digested with EcoRI and PstI enzymes and subsequently ligated with a chloramphenicol resistant plasmid backbone (Cm). Transforming bacteria were created with this plasmid and seeded on LB-Agar+Cm plates. After growth, colonies were selected based on their color (white) and DNA extraction was performed using the Promega PureYield Plasmid Miniprep System kit. The resulting DNA is used for further digestion with EcoRI and PstI. The digests are then run on a 0.75% agarose gel at 90 mV voltage and constant amperage. BioRad brand RedSafe is used as an intercalating developing agent.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NotI site found at 554
Illegal NotI site found at 1698 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 268
Illegal BglII site found at 1611
Illegal BamHI site found at 747
Illegal XhoI site found at 1109
Illegal XhoI site found at 1358 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 889
Illegal NgoMIV site found at 1814 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 793
Contribution
- Group: [1]
- Author: Molina Calvo, Alonso
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