Part:BBa_K823044
MazF a bacterial toxin
MazF is a bacterial toxin from E. coli and degrades mRNA
Our team used this part in the combination with PydfG,ecf41Bli aa 1-204,PspoIVB/PsspK in the [http://2012.igem.org/Team:LMU-Munich/Germination_Stop#Suicide_switch Suicide switch].
σ G, the last sigma factor in the forespore, activates PspoIVB, producing ECF41 during Sporulation. This then activates the PydfG promoter producing MazF, theoretically killing cells after germination.
Characterization of mazE&mazF by team BUCT 2021:
We designed the part(BBa_K3875026) to characterize mazE and mazF.
We performed experiment to work our final genetic circuits and characterized mazE and mazF.
This experiment consists of the four parts below:
1.Obtain the mazEF gene and the phyb gene
2.Build a gene expression vector
3.Import the gene expression vector to detect the properties of engineered bacteria
1.Methods:
1.Design primers with a homologous arm, using pcr to obtain the target genes mazEF and phyb from E. coli BW25113, the pcr program is shown in the figure
Figure1.Pcr program:
Figure. Obtain mazE and mazF gene：
2. Connecting five gene fragments using gibson assembly method
Figure. Gibson program
3. Verify the length of the expression vector
Figure. Verify the length of the expression vector
Figure. Enzyme cut validation results
4.The expression carrier is imported into the Nissle 1917 receptor state and cultured under oxygen-free conditions to culture 48h
Figure .The engineered bacteria are cultured under oxygen-free conditions:
5. Observe the results and verify them
2. Results
Cultured 16h under aerobic conditions, no colonies were found to grow, which proved that there was no escape of engineering bacteria under aerobic conditions, which is in good compliance with the safety principle.
Figure．Aerobic conditions cultured 16h, engineering bacteria died:
After constantly groping for reaction conditions, it was found that Nissle 1917 grew very slowly in an oxygen-free environment, requiring 48-72h growth in order to see the obvious colony of the crucible in the aerobic air environment, it is not possible to observe obvious traces of engineering bacteria growth, which can indicate the success of the suicide system.
Figure. The culture contrast between aerobic environment and anaerobic environment:
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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