Coding
mazF, ydcE

Part:BBa_K3796203

Designed by: Yang Feiyue   Group: iGEM21_CAU_China   (2021-09-23)
Revision as of 08:32, 14 October 2021 by Chouuken (Talk | contribs) (Characterization)


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 inserted at downstream of lac operon whose lacI was under 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 of ndoA. CFU assay was carried out to characterize the killing effect of ndoA instead of determining OD600 in order to get rid of dead bacterial cells. We added 0.8mM IPTG as OD600 reached 0.6, and estimate cfu by spreading some of the culture on 3 LB solid medium every hour. Cfu was quantified by counting the colonies on one plate and normalizing the number to volume of 1 mL culture. C. glumaticum carrying empty vector (IPTG added and not added) and C. glutamicum carrying our clone (non-induction) were dealt with in the same way as control.

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It is visually discovered that the number of colonies carrying lac operon and ndoA fell off in the presence of IPTG, while the same bacteria grew well within the first four hours and then died due to the leakiness. Now we clearly know that toxin ndoA works well in C. glutamicum.


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]


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Categories
Parameters
None