Coding

Part:BBa_K302033

Designed by: Philip Hall   Group: iGEM10_Newcastle   (2010-10-25)
Revision as of 10:13, 20 October 2019 by Fz903 (Talk | contribs) (Characterized by BNU-China 2019)

mazF

Encodes a stable non-specific ribonuclease in Bacillus Subtilis. It is used in conjungtion with mazE in Bacillus Subtilis to provide a toxin-antitoxin kill switch in various stressful conditions. When expression of both genes is turned off (as both are under contol of same promoter) mazE will be degraded faster than mazF. There is then no inhibiton of mazF, killing the cell.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Characterized by BNU-China 2019

We characterize mazF (BBa_K302033) by an induced suicide system, in which the downstream mazF (BBa_K302033) gene encoding toxin MazF is put under the control of L-arabinose induced promoter pBAD (BBa_K206000). MazF is an endoribonuclease that cleaves RNAs at ACA sites and causes the death of microbe as a reporter [1]. As a result, we can characterize mazF in a cell density-dependent manner in Escherichia coli K-12.

2019 BNU-China BBa K3036005 change.png

In order to characterize the toxicity of protein encoded by mazF, we take engineered microbe without induction as control group. As is shown in Fig.1, the cell number of experimental groups show a significant decrease upon induction, which indicates the protein encoded by mazF is lethal to E. coli.

2019 BNU-China BBa K206000 pic2.png

Figure 1 Cell number declines after induction. It proves that protein encoded by mazF is lethal to E. coli.

Furthermore, in order to prove that MazF 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 numbers of alive cells differ. Hence, we reach a conclusion that protein encoded by mazF mediates suicide of cells without causing lysis of bacterial cells.

2019 BNU-China BBa K302033 change.jpg

Figure 2 Absorbance at 600nm with time.

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 10^7 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 cm^2 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.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


[edit]
Categories
//cds
//chassis/prokaryote/bsubtilis
Parameters
None