Part:BBa_K3198001

HicB

This part contains the antitoxin component of a type II toxin-antitoxin (TA) system. It functions as an mRNA interferase antitoxin; overexpression prevents HicA-mediated cessation of cell growth and cell death. HicB functions as an mRNA interferase antitoxin. Expression of HicB (145 aa) prevented HicA-mediated inhibition of cell growth. HicB neutralizes HicA and therefore functions as an antitoxin. HicB could resuscitate cells inhibited by HicA.

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
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 48
  Illegal BsaI.rc site found at 88
  

Usage

HicB functions as an mRNA interferase antitoxin. Expression of HicB (145 aa) prevented HicA-mediated inhibition of cell growth. HicB neutralizes HicA and therefore functions as an antitoxin. HicB could resuscitate cells inhibited by HicA.

Biology

HicB is from hicAB locus of Escherichia coli K-12.

Characterisation

Team NUS 2019 has added another new biobrick (BBa_K3198001) into iGEM repository this year. This biobrick was found to possess the ability to neutralize the effect of HicA( biobrick id) and therefore functions as an antitoxin. For this reason, team NUS 2019 used this biobrick as part of their sleep-wake module to control the growth of E. coli in their project.

(BBa_K3198001) was placed under an arabinose-inducible promoter and various arabinose concentrations were used to determine their ability to resume cell growth native MG1655.

Characterization of cells transformed with this plasmid was performed at 37°C for 12h continuously. Both toxin and antitoxin inducers were added together at 0h, with IPTG concentration fixed at 500uM with varying arabinose concentrations of 0M, 0.0266M, 0.133M and 0.266M.

After the addition of 500μM IPTG into cells co-transformed with (BBa_K3198000) and (BBa_K3198001) plasmid, it was shown that even at the lowest arabinose concentration of 0.0266M, the growth of the cells was able to restore to the same level as uninduced cells. On the other hand, cells only induced with 500μM IPTG did not managed to show growth resumption in the absence of arabinose. This demonstrated the function of (BBa_K3198001) as an antitoxin capable of neutralising (BBa_K3198000) effect on native MG1655 growth.

figures

At 0uM IPTG, all the four curves have similar trend, indicating that when toxin is not induced, there is little effect from the induction of antitoxin.

figure

The combined graph shows that when toxin is induced at 500μM IPTG, at 0M arabinose, final OD plateau at 0.8, indicating no resumption of cell growth. At its optimal arabinose concentration of 0.0266M, cell growth resumes and continues to a level greater than 1.6 OD.

References

Jorgensen, M. G., Pandey, D. P., Jaskolska, M., & Gerdes, K. (2008). HicA of Escherichia coli Defines a Novel Family of Translation-Independent mRNA Interferases in Bacteria and Archaea. Journal of Bacteriology, 191(4), 1191–1199. doi: 10.1128/jb.01013-08

Maisonneuve, E., Shakespeare, L. J., Jørgensen, M. G., & Gerdes, K. (2011). Bacterial persistence by RNA endonucleases. Proceedings of the National Academy of Sciences, 108(32), 13206–13211. doi: 10.1073/pnas.1100186108