Part:BBa_K1321333:Experience

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Applications of BBa_K1321333

This part was characterised in combination with BBa_K1321334, and the resulting part was submitted as BBa_K1321336. BBa_K1321333 and BBa_K1321334 were cloned by Biobrick cloning, by linearising BBa_K1321333 with SpeI and PstI, and later ligating BBa_K1321334, previously digested with XbaI and PstI to generate a front insert, at a 1:1 ratio using T4 ligase. The ligation mix was transformed into chemically competent DH10B Escherichia coli and plated on LB Agar+Chloramphenicol plates, then incubated overnight at 37 degrees. 50 mL Falcon tubes containing 5 ml LB supplied with 50 ug/ml Chloramphenicol were inoculated with a selection of freshly grown colonies for further restriction analysis and gene sequencing verification.

BBa_K1321336 was characterised in conjunction to an additional pLAC-inducible expression system containing AcsC and AcsD (optimised coding sequences submitted as Part 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.

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.

Figure 1 - Congo Red assay, positive colonies

Figure 2 - Congo Red assay, negative control

Whilst the AcsC and AcsD elements of the cellulose synthesis operon (coded in part BBa_K1321335) are needed for cellulose secretion into the extracellular space, we were 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.

Figure 3 - Assaying Congo Red (CR) binding by measuring the changes in absorbance at 490nm

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.

Figure 4 - Assaying Congo Red (CR) binding by measuring the changes in absorbance at 490nm

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.

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