Difference between revisions of "Part:BBa K3796203"
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===Usage and Biology=== | ===Usage and Biology=== | ||
+ | MazF is an mRNA interferase which cleaves mRNAs at a specific site, and <i>ndoA</i>, also called <i>ydcE</i> or <i>mazF-bs</i> shows a similar activity according to references given below. <i>ndoA</i> belongs to a type Ⅱ toxin-antitoxin system in <i>Bacillus subtilis</i>. There is great possibility that no antitoxin exists in <i>C. glutamicum</i> to inhibit EndoA encoded by <i>ndoA</i>. So it can be used to kill engineered <i>C. glutamicum</i> at a proper time. | ||
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+ | ===Characterization=== | ||
+ | This year, our team designed a kill switch in <i>C. glutamicum</i> using <i>ndoA</i> from <i>Bacillus subtilis</i> subsp. subtilis str. 168 under the control of PgsiB (alkali inducible promoter) and Patp2 (alkali inducible promoter). We aimed to test if the over-expression of <i>ndoA</i> in <i>C. glutamicum</i> can kill the engineered bacteria effectively, and documented our works and failures in iGEM Parts to inspire future iGEM teams for kill switch design. | ||
+ | To verify that <i>ndoA</i> has function of killing <i>C. glutamicum</i>, <i>ndoA</i> was inserted into PXMJ19 to construct a simple circuit with tac promotor, lac operon, <i>ndoA</i> and terminator rrnB in order.<br> | ||
+ | To design a quick and visual way, ‘divided plate’ assay was carried out at the beginning. <br> | ||
+ | Bacteria carrying empty vector was added into 1 Ep tube and bacteria carrying Ptac/<i>lacIq-ndoA</i> circuit was added into 2 Ep tubes with 1.8 mL LB liquid containing 10 μg/mL chloramphenicol respectively. Incubate the culture in a shaker at 30 °C, 220 rpm until OD<sub>600</sub> reached 0.6. | ||
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+ | CFU assay was carried out to characterize the killing effect of <i>ndoA</i> instead of determining OD<sub>600</sub> to get rid of dead bacterial cells. | ||
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Revision as of 12:49, 12 October 2021
ndoA (endoribonuclease toxin in Bacillus subtilis)
This coding region ndoA(also mazF, ydcE) encodes endoribonuclease EndoA, a toxic component of a type II toxin-antitoxin (TA) system. It encodes endoribonuclease toxin in Bacillus subtilis that is a UACAU‐specific mRNA interferase, cleavaging after the first U. Previous research has proved that its overexpression is toxic for cell growth and it functions as normal in Escherichia coli.
Usage and Biology
MazF is an mRNA interferase which cleaves mRNAs at a specific site, and ndoA, also called ydcE or mazF-bs shows a similar activity according to references given below. ndoA belongs to a type Ⅱ toxin-antitoxin system in Bacillus subtilis. There is great possibility that no antitoxin exists in C. glutamicum to inhibit EndoA encoded by ndoA. So it can be used to kill engineered C. glutamicum at a proper time.
Characterization
This year, our team designed a kill switch in C. glutamicum using ndoA from Bacillus subtilis subsp. subtilis str. 168 under the control of PgsiB (alkali inducible promoter) and Patp2 (alkali inducible promoter). We aimed to test if the over-expression of ndoA in C. glutamicum can kill the engineered bacteria effectively, and documented our works and failures in iGEM Parts to inspire future iGEM teams for kill switch design.
To verify that ndoA has function of killing C. glutamicum, ndoA was inserted into PXMJ19 to construct a simple circuit with tac promotor, lac operon, ndoA and terminator rrnB in order.
To design a quick and visual way, ‘divided plate’ assay was carried out at the beginning.
Bacteria carrying empty vector was added into 1 Ep tube and bacteria carrying Ptac/lacIq-ndoA circuit was added into 2 Ep tubes with 1.8 mL LB liquid containing 10 μg/mL chloramphenicol respectively. Incubate the culture in a shaker at 30 °C, 220 rpm until OD600 reached 0.6.
CFU assay was carried out to characterize the killing effect of ndoA instead of determining OD600 to get rid of dead bacterial cells.
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]