Part:BBa_K3198001

HicB

This part contains the antitoxin component of a type II toxin-antitoxin (TA) system.

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
  

Description

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. Refer to our wiki https://2019.igem.org/Team:NUS_Singapore/Design#Characterization for more details.

Usage

Team NUS Singapore 2019 has added a new biobrick (BBa_K3198001) into the iGEM repository this year. This biobrick was found to possess the ability to neutralize the effect of BBa_K3198000 and therefore functions as an antitoxin. For this reason, team NUS Singapore 2019 used this biobrick as part of their sleep-wake module to control the growth of E. coli. More specifically, to overcome the pre-induced dormant state of these cells.

Biology

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

Characterization

Since BBa_K3198001 has the ability to neutralize BBa_K3198000 in nature, team NUS Singapore 2019 hypothesized that the induction of both BBa_K3198001 and BBa_K3198000 would result in a growth curve that is comparable to that of control MG1655 which does not possess any plasmid.

To test this hypothesis, BBa_K3198001 was placed under an arabinose-inducible promoter and various arabinose concentrations were used to determine their ability to resume growth in 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 and arabinose concentration fixed at 500μM and 26.6mM respectively.

After the addition of 500μM IPTG into cells co-transformed with BBa_K3198000 and BBa_K3198001 plasmid, it was shown that the cells were able to restore their growth back to the same level as control MG1655 (Figure 2). On the other hand, cells induced with 500μM IPTG only did not manage to show growth resumption in the absence of arabinose (Figure 1).


Figure 1: Growth curve of control MG1655 unaffected by the addition of IPTG while MG1655 co-transformed with both plasmids observed a reduction in growth upon the addition of IPTG.


Figure 2: MG1655 co-transformed with both plasmids demonstrated a normal growth curve which is comparable to control MG1655 when arabinose concentration of 26.6mM was added together with IPTG.

To ensure that the comparison of cell growth between control MG1655 and MG1655 co-transformed with both plasmids is appropriate, the possible effect of IPTG and arabinose on the growth of control MG1655 was investigated. No significant effect was observed in both strains (Figure 3).


Figure 3: Growth curve of control MG1655 when treated with various inducers.

Taken together, these results demonstrate that BBa_K3198001 does indeed function as an antitoxin capable of neutralising BBa_K3198000 effect on native MG1655 growth.

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

Sources

BBa_K3198001 originated from E. coli K12 and its sequence was synthesized by IDT.