Difference between revisions of "Part:BBa K3036005"

Latest revision as of 03:03, 19 October 2021

Arabinose-induced suicide switch

This arabinose-induced suicide switch consists of pBAD (BBa_K206000) and mazF (BBa_K302033), aiming to enable users of this microbe to terminate engineered bacteria inside their intestines with L-arabinose whenever needed. This suicide switch does no harm to human and can be used by direct in-taking of inducer. PBAD (BBa_K206000) is applied for heterologous gene expression due to its advantages, including moderately high expression levels, induction by a low-cost and non-toxic monosaccharide L-arabinose and tight regulation of transcription, which is particularly significant to expressing toxins [1]. MazF is an endoribonuclease that cleaves RNAs at ACA sites and causes the death of microbe, mediates suicide of cells without causing lysis of bacterial cells[2].

Biology and Usage

This arabinose-induced suicide switch consists of pBAD (BBa_K206000) and mazF (BBa_K302033), aiming to enable users of this microbe to terminate engineered bacteria inside their intestines with L-arabinose whenever needed. This suicide switch does no harm to human and can be used by direct in-taking of inducer. PBAD (BBa_K206000) is applied for heterologous gene expression due to its advantages, including moderately high expression levels, induction by a low-cost and non-toxic monosaccharide L-arabinose and tight regulation of transcription, which is particularly significant to expressing toxins [1]. MazF is an endoribonuclease that cleaves RNAs at ACA sites and causes the death of microbe, mediates suicide of cells without causing lysis of bacterial cells. [2]

Sequence and Features

Design and Properties

Arabinose-induced suicide switch is characterized in a system where the mazF (BBa_K302033) gene encoding toxin is put under the control of L-arabinose induced promoter pBAD (BBa_K206000).

As is shown in Fig.1, the cell number of experimental groups show a significant decrease upon induction, which indicates the arabinose-induced suicide switch works upon induction of 1.25μM/L and 2.5μM/L L-arabinose.

Then, in order to prove that this arabinose-induced suicide switch kills the cell without lysing it, we measure OD600 of each sample to give an overall number of intact bacteria, dead and alive. As is shown in Fig.2, there is little difference between control and experimental groups, although it is validated that numbers of alive cells differ. Hence, we reach a conclusion that the switch mediates suicide of cells without causing lysis of bacterial cells, which is harmless to native microbe.

Experimental approach

1.Transform the plasmids into E. coli DH5α competent cells.
2.The engineered bacteria are cultured in 200mL LB-ampicillin (50 ng/µl) medium overnight at 37℃, 200rpm;
3.Equally divide the culture into 90 centrifuge tubes, which is 1mL respectively. Centrifuge them at 4000rpm for 5 minutes. Discard the liquid.
4.Resuspend 30 tubes of collected bacteria with LB-ampicillin (50 ng/µl) containing 1.25μM/L and 2.5μM/L L-arabinose respectively as experimental groups. Resuspend 30 tubes of bacteria with pure LB-ampicillin (50 ng/µl) medium.
5.Collect 3 tubes of all groups every 6 hours, dilute all of the samples to 107 times and then spread them on solid LB-ampicillin (50 ng/µl) medium separately. At the same time, refresh the medium to maintain the concentration of L-arabinose.
6.Count the number of colonies in 5 cm2 per plate after cultured for 24 hours at 37℃
7.Three repicas are tested in each group.

Reference

[1] Diana Széliová, Ján Krahulec, Martin Šafránek, et al. Modulation of heterologous expression from PBAD promoter in Escherichia coli production strains[J]. Journal of Biotechnology, 2016, 236:1-9.
[2] Nigam A, Ziv T, Oron-Gottesman A, Engelberg-Kulka H2019. Stress-induced MazF-mediated proteins in Escherichia coli. mBio 10: e00340-19. doi:10.1128/mBio.00340-19.

BNUZ-China 2021

We used two different concentrations of arabinose in our experiment. After induction, the survival rate of engineered bacteria in the experimental group decreased significantly. Meanwhile, the OD600 of the culture also decreased, indicating that high concentrations of arabinose induced bacterial lysis.

Live cells are counted over time after induction, and the number is significantly reduced under the effect of MazF expression (Figure 3). The OD600 of the experimental group decreased significantly, which proved that suicide induced by high concentration of arabinose would cause cell lysis (Figure 4).

Figure 3. Viable cell count changes over time

Figure 4. OD600 change over time at different L-arabinose concentrations