Coding
mazF, ydcE

Part:BBa_K3796203

Designed by: Yang Feiyue   Group: iGEM21_CAU_China   (2021-09-23)
Revision as of 17:43, 11 October 2021 by Chouuken (Talk | contribs) (→‎Design Page)


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.


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]


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.

CFU assay was carried out to determine the effect of ndoA. Three groups of plates, empty vector (with IPTG), Ptac/lacIq-ndoA (non-induction) and Ptac/lacIq-ndoA (induction) were used. CFU of three groups above is determined every one hour to catch the killing process. At first, LB solid medium was used in each plate for these three groups. However, there was no significant difference between the last two group. We assume that there must be leakiness of Ptac/lac operon in C. glutamicum. So, we used LBG medium to replace LB medium of Ptac/lacIq-ndoA (without induction) in order to repress the expression of ndoA when no IPTG exists. It was discovered that the number of colonies carrying lac operon and ndoA fell at presence of IPTG, while the same bacteria without IPTG grew well at first and died due to leakiness after 4 hours.

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In the assay, we noticed that toxin gene ndoA resulted in a very slow colony growth, which was indirect evidence.

What’s more, ndoA couldn’t be detected by the method of PCR as time went in the CFU assay, which cut the contrast.

To give a sharp view of toxin ndoA and avoid leakiness, another assay was also carried out by dividing a plate into four parts equally and used LBG medium in one part, LB in the others. Empty vector (with IPTG), empty vector (without IPTG) and Ptac/lacIq-ndoA (induction) were grown respectively on LB parts, Ptac/lacIq-ndoA (non-induction) was grown on the LBG part. 

//image

As you can see, the number of colonies of C. glutamicum carrying ndoA will can be much smaller because of the presence of IPTG.

Design Page

Design

In the very beginning, ndoA from B. Subtilis, mazF from E. Coli and ndoA from C. Glutamicum were considered to be the toxin gene in our kill switch. However, ndoA stood out for two reasons. On one hand, its annotation score is 5 on the Uniprot website. On the other hand, B. Subtilis is Gram-positive bacteria just like C. Glutamicum.

Build

We have only constructed a simple genetic circuit for ndoA test so far, which is composed with Ptac, ndoA and rrnB T1&T2 using ClonExpress II one-step cloning kit (Vazyme Biotech, China). This expression clone is transformed into DH5α first and then into C. glutamicum by Electroporation.

Test

We test our clone in several ways, including agarose gel electrophoresis, digestion with restriction endonuclease and colony PCR to assure our clones correct and ready to work.

References

[1]Pellegrini O, Mathy N, Gogos A, Shapiro L, Condon C. The Bacillus subtilis ydcDE operon encodes an endoribonuclease of the MazF/PemK family and its inhibitor. Mol Microbiol. 2005;56(5):1139-1148. doi:10.1111/j.1365-2958.2005.04606.x
[2]Park JH, Yamaguchi Y, Inouye M. Bacillus subtilis MazF-bs (EndoA) is a UACAU-specific mRNA interferase. FEBS Lett. 2011;585(15):2526-2532. doi:10.1016/j.febslet.2011.07.008
[3]Ishida Y, Park JH, Mao L, Yamaguchi Y, Inouye M. Replacement of all arginine residues with canavanine in MazF-bs mRNA interferase changes its specificity. J Biol Chem. 2013;288(11):7564-7571. doi:10.1074/jbc.M112.434969
[4]Simanshu DK, Yamaguchi Y, Park JH, Inouye M, Patel DJ. Structural basis of mRNA recognition and cleavage by toxin MazF and its regulation by antitoxin MazE in Bacillus subtilis. Mol Cell. 2013;52(3):447-458. doi:10.1016/j.molcel.2013.09.006

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