Part:BBa_K3796203

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. After incubating the culture in a shaker at 30 °C, 220 rpm until OD₆₀₀ reached 0.6, we prepared LB plates whose each quarter had been marked A,B,C and D. In detail, IPTG was spread on Quarter A & B ,and bacteria carrying empty vector was spread on Quarter A & C, then bacteria carrying our clone was spread on Quarter B & D. All the plates were incubated at 30 °C for a certain time.

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It gave a great contrast between Quarter B & D, and there were much fewer colonies than Quarter A & C, which verified ndoA kills C. glutamicum strongly. We assume that ndoA killed C. glutamicum due to leakiness Then

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

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]