Part:BBa_K1336005:Experience

Antisense for ispB

The effects of the presence of this part (no promoter) on E. coli growth were tested. This was carried out by measuring bacterial OD at regular intervals of 1 hour, in the different media. Each of the tubes from which the samples were extracted contained initially 10mL of LB medium (formulated by mixing 25 gr of Sigma-Aldrich L3522 Luria broth with 1 L of Milli-Q water). Apart from the plasmid-free controls, each tube also contained 10uL of 25ng/uL Chlorampehicol. The cells used were Invitrogen™ DH5α™, which show the following genotype: F– Φ80lacZΔM15 Δ(lacZYA-argF) U169 recA1 endA1 hsdR17 (rK–, mK+) phoA supE44 λ– thi-1 gyrA96 relA1. All cultures were grown at 37 ºC and shaking at 250rpm.

To these tubes, 100uL of the three different concentrations for each of the two dyes were added, to give the desired final concentrations as specified below. To the controls, 100uL of sterile water was added. They were then incubated for the time frames indicated in the figures below, and at the specified time points two samples of 200uL were taken into two cuvettes to then be diluted into 1.8mL of LB (from the same batch as that found in the culture tubes). The absorbance shown on the graphs is the absolute value, not the dilution. Readings were taken in a standard spectrophotometer at 680nm; the choice of wavelength aims to reduce to a minimum the interference caused by the strong absorption of the dyes, while still measuring bacterial density. Although high-concentration RB5 still shows an absorption much higher than the other samples, the curve is preserved and so it allows to analyse how the presence of dyes might interfere with bacterial growth. The full protocol for this assay can be found here.

Figure 1a - BBa_K1336005 ispB xenobiological module is compatible with Reactive Black 5 dye-contaminated waste waters. Graph showing that E.coli transformed with BBa_K1336005 ispB shows comparable growth to the plasmid-free control in LB media with RB5 dye. OD measured at 680nm and Time is shown in hours after incubation. Error bars indicate SEM, n=2.

Figure 1b - BBa_K1336005 ispB xenobiological module is compatible with Acid Orange 7 dye-contaminated waste waters. Graph showing that E.coli transformed with BBa_K1336005 ispB shows comparable growth to the plasmid-free control in LB media with AO7 dye. OD measured at 680nm and Time is shown in hours after incubation. Error bars indicate SEM, n=2.

As expected, the sole uninduced presence of BBa_K1336005 in transformed cells seems to have no negative effect on E. coli growth, since the growth of the transformed cells is comparable to that of the plasmid-free control. In addition to this, the presence of azo-dyes AO7 or RB5 in the medium at the indicated concentrations doesn't seem to disturb the growth of BBa_K1336005-transformed cells either. This shows that the non-induced part would be compatible with the viability of E. coli also in az-dye contaminated media.

A functional device for this part using a Lac-induced promoter was characterised here

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