Part:BBa_K1321336
AraC-pBAD-AcsAB
This composite part is the result of combining parts BBa_K1321333 and BBa_K1321334 by BioBrick cloning. It contains the Arabinose-inducible promoter pBAD and also codes for the transcriptional inhibitor/activator AraC. Furthermore, pBAD is followed by the AcsAB open reading frame, which in turn codes for the cellulose synthase enzyme, key player for cellulose production.
Usage and Biology
BBa_K1321336 was characterised both on its own and in conjunction to an additional pLAC-inducible expression system containing AcsC and AcsD (derived from BBa_K1321335) cloned into a medium-to-low copy number plasmid, pSB3K3. The functions of AcsC and AcsD are yet not very well known but are believed to play a crucial role in cellulose crystallisation and secretion into the extracellular space. In the presence of AcsC and AcsD, cellulose production was assayed by plating transformed cells on Congo Red assay plates containing 20uM CR, 0.5mM IPTG, 0.1% Arabinose, 1% Glucose, 25ug/ml Chloramphenicol and 25ug/ml Kanamycin. Cellulose-producing E.coli colonies turned red in the presence of CR.
Whilst the AcsC and AcsD elements of the cellulose synthesis operon (coded in part BBa_K1321335) are needed for cellulose release, we were also interested in exploring whether AcsAB only would be enough to produce the polymer in E.coli. The functionality of part BBa_K1321336 was assayed by inducing the system with 0.1% Arabinose in 5mL LB supplied with 1% Glucose and Chloramphenicol. Overnight incubations were set up at 30 °C and 37 °C, and both empty vector controls and un-induced controls were also assayed for cellulose production.
During sonication, LB, soluble and non-soluble fractions were produced and kept for further analysis. Congo Red (CR) at a concentration of 20uM was added to all samples, which were then incubated for 2 hours at room temperature and static conditions, prior to absorbance measurements being taken at 490nm. By subtracting the absorbance values of the samples from the absorbance value of a PBS+CR control, it is possible to qualitatively assay the shift in spectral properties of the diazo dye, driven by CR binding to the cellulose fibres.
Because AcsC and AcsD were absent, the LB and soluble fractions derived from sonication were expected to contain no cellulose; the main reason being due to the inability of the cells to extrude the fibers, in addition to cellulose being highly hydrophobic hence should precipitate together with the remaining immiscible cellular elements released during sonication. As predicted, no spectral changes were reported in the LB (figure 3) and soluble fractions (data not shown), however these were observed on non-soluble samples (figure 4) derived from cultures grown at 30 degrees and static conditions (figure 2). These results, supported with two biological repeats and technical triplicates, suggest that BBa_K1321336 is functional and able to produce some cellulose at 30 degrees, in the absence of AcsC and AcsD.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1205
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 3049
Illegal BglII site found at 3763
Illegal BglII site found at 5686
Illegal BamHI site found at 1144
Illegal BamHI site found at 2059
Illegal BamHI site found at 5053 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 979
Illegal AgeI site found at 3035
Illegal AgeI site found at 3112
Illegal AgeI site found at 3668
Illegal AgeI site found at 4469
Illegal AgeI site found at 4561
Illegal AgeI site found at 4667 - 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI site found at 961
| None |