Part:BBa_K1336006:Experience

LEC + Antisense ispB

The effects of uninduced BBa_K1336006 on cell growth on different media was tested, starting with plain LB and azo-dyes AO7 and RB5. 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 1 - BBa_K1336006 LEC-ispB xenobiological module is compatible with Reactive Black 5 and Acid Orange 7 dye-contaminated waste waters. Graph showing that E.coli transformed with BBa_K1336006 ispB shows comparable growth to the plasmid-free control in LB media with AO7 and RB5 dyes. OD measured at 680nm and Time is shown in hours after incubation. Error bars indicate SEM, n=2.

The part sequence showed not to be toxic for E. coli in the media and conditions tested. The next step was to test if the induction of the part with IPTG compromised bacterial growth in LB. For this, tubes were set up as above, but 10uL of 1M IPTG were added to the tubes in which induction ofthe device would take place.

Figure 2 - Growth of BBa_K1336006-transformed cells in LB media shows indicative decrease after IPTG induction Graph showing growth of E. coli transformed with BBa_K1336006 and the effect of IPTG induction, using BBa_K1336005 as a control. . OD measured at 600nm and Time is shown in hours after incubation. Error bars indicate SEM, n=2.

After having tested the inhibition of growth produced by the induction of BBa_K1336006 in plain LB, the next step was to study the viability of cells expressing this part in minimal media, in order to observe whether growth on more selective media would make the cells more susceptible to the effects of BBa_K1336006. Both M9 and CAS-containing-M9 media were prepared according to the protocol available in opennetware.org;
-1x M9 salts
-2mM MgSO4
-0.1mM CaCl2
-0.4% glucose (after autoclaving)
-CAS-amino acids (in 1 of the media only)
-In sterile H2O

The media was distributed in the Falcon tubes along with the cells in an indentical fashion as describe above for the LB. The measurements were also taken in the same way.

Figure 3a - Growth of BBa_K1336005 ispB and BBa_K1336006 LEC-ispB xenobiological module in M9 minimal media Graph showing growth of E. coli transformed with BBa_K1336006 and the effect of IPTG induction, using the part only (without lacI) as a control. . OD measured at 680nm and Time is shown in hours after incubation. Error bars indicate SEM, n=2.

Figure 3b - Growth of BBa_K1336005 ispB and BBa_K1336006 LEC-ispB xenobiological module in M9+CAS minimal media Graph showing Graph showing growth of E. coli transformed with BBa_K1336006 and the effect of IPTG induction, using the part only (without lacI) as a control. Minimal media with addition of casamino acids was used to comfirm the auxotrophies of the strain we used. . OD measured at 680nm and Time is shown in hours after incubation. Error bars indicate SEM, n=2.

The ispB antisense RNA silencing tool hasn't proved effective in our initial research in LB media. We decided to characterise the part in M9 minimal media to confirm that quinones were not externally provided in LB media, which would detriment the usefulness of a knock-out of quinones biosynthesis. We are in the phase of development of further silencing tools, in collaboration with the Edimburgh team in order to test if we can complement ubiquinone and menaquinone with synthetic equivalents after silencing ispB the key gene for their biosynthesis.

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