Difference between revisions of "Part:BBa K3198008"

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pBAD-HicB

This part contains the arabinose-inducible promoter pBAD and antitoxin component HicB 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.

Description

This part contains the arabinose-inducible promoter pBAD and antitoxin component HicB 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#Demonstration for more details.

Usage

Team NUS Singapore 2019 has added a new biobrick (BBa_K3198008) into the iGEM repository this year. This biobrick was found to possess the ability to neutralize the effect of HicA 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 Escherichia coli K-12. More specifically, to overcome the pre-induced dormant state of these cells. Coupled to BBa_K3198007, we were able to demonstrate prolonged functional lifespan in 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 HicB has the ability to neutralize HicA in nature, team NUS Singapore 2019 hypothesized that the induction of both HicA and HicB would result in a growth curve that is comparable to that of uninduced MG1655.

Having showed that the induction of BBa_K3198001 expression restores growth and protein production previously arrested by BBa_K3198000, team NUS Singapore 2019 went on further to demonstrate the functionality of their parts using bioluminescence production as a proof-of-concept. They reasoned that since BBa_K3198000 and BBa_K3198001 were able to regulate constitutive protein production, the parts should work the same in inducible protein production systems. Most importantly, the team hypothesized that cells expressing both BBa_K3198000 and BBa_K3198001 should stay viable over a longer period of time to still be able to produce protein compared to uninduced control cells.

To test this hypothesis, composite part BBa_K3198008 containing HicB antitoxin under the control of arabinose-inducible promoter is constructed. Coupled with another composite part BBa_K3198007 containing HicA toxin (BBa_K3198000) and LuxCDABE gene (BBa_K325909), the team co-transformed both plasmids into MG1655 and grew the cells in falcon tubes at 37°C.


Figure 1: Plasmid map showing HicA under the control of lac-inducible promoter while LuxABCDE is under the control of arabinose-inducible promoter.

The cells were grown in the shaking incubator for up to 10 days, with 200μL culture aliquoted into microplate and induced with arabinose for lux production on day 10. The microplate was loaded into a microplate reader and cells were measured for OD₆₀₀ and luminescence at 29°C for 12h continuously.

The results showed that on day 10, growth-arrested cells expressing HicA observed a higher level of luminescence production compared to uninduced control cells after both samples were added with arabinose (Fig 2). To put it simpler, while uninduced cells lost their ability to produce protein on day 10, the growth-arrested cells retained the ability to produce sufficient amount of luminescence when woken up by the expression of HicB antitoxin which was also induced by arabinose.


Figure 2: Characterization of cells on day 10. Growth curve of control and IPTG-treated MG1655 upon the addition of 2% arabinose right at the beginning.


Figure 3: Characterization of cells on day 10. Total luminescence graph of control and IPTG-treated MG1655 upon the addition of 2% arabinose right at the beginning.

Taken together, we are able to demonstrate growth-arrested cells ability to stay viable and functional for a longer period of time compared to uninduced cells without the expression of BBa_K3198007 and BBa_K3198008. We hypothesized that when growth is arrested, the cells managed to conserve cellular reserves such as ATP as supported by Lobritz et al (2015) and as a result, prolong the functional viability of cells until protein production is induced. This demonstration opens up various applications that can benefit from our growth switch, ranging from controllable bioluminescence production as a form of light source to therapeutic biosensors that can remain in the body for a long period of time until needed to perform its function.

References

Zhang, Y., Zhang, J., Hoeflich, K.P., Ikura, M., Qing, G. and Inouye, M. (2003) MazF cleaves cellular mRNAs specifically at ACA to block protein synthesis in Escherichia coli. Molecular Cell, 12, 913–923.

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

Lobritza, M.A., Belenky, P., Porterb, C.B.M., Gutierrezb, A., Yang, J.H., Schwarzg, E.G., Dwyerh, D.J., Khalila,A.S., & Collins, J.J. (2015). Antibiotic efficacy is linked to bacterial cellular respiration. Proceedings of the National Academy of Sciences, 112(27), 8173–8180. doi:10.1073/pnas.1509743112

Source

BBa_K3198001 originated from Escherichia coli K-12 and its sequence was synthesized by IDT.

Design Considerations

Sequence and Features