Part:BBa_K3198007
HicA-LuxABCDE
This part contains the toxin component of a type II toxin-antitoxin (TA) system. HicA is a probable translation-independent mRNA interferase. Bacterial lux operons encodes five enzymes involved in the light-generating pathway. LuxA and LuxB encode the two subunits of the bacterial luciferase, while the products of LuxC, LuxD and LuxE synthesise the substrate for the light emitting reaction, tetradecanal. The exact function of LuxG is unknown, and it appears to be non-essential for light emission, but its presence increases light output.
Description
This part contains the toxin component of a type II toxin-antitoxin (TA) system. HicA is a probable translation-independent mRNA interferase.
Bacterial lux operons encodes five enzymes involved in the light-generating pathway. LuxA and LuxB encode the two subunits of the bacterial luciferase, while the products of LuxC, LuxD and LuxE synthesise the substrate for the light emitting reaction, tetradecanal. The exact function of LuxG is unknown, and it appears to be non-essential for light emission, but its presence increases light output.
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_K3198007) into the iGEM repository this year. This biobrick was found to possess bacteriostatic effect as reported by Gerdes et al in 2008 and was therefore used by team NUS Singapore 2019 as part of their sleep-wake module to control the growth of Escherichia coli K-12 by inducing dormancy in these cells. Coupled to the lux operon, we used this biobrick to demonstrate prolonged functional lifespan in these cells.
Biology
HicA originates from the HicAB locus of Escherichia coli K-12. HicA toxins cleave mRNAs independently of the ribosome. Overexpression leads to cleavage of a number of mRNAs and tmRNA, in a translation-independent fashion, suggesting that HicA is an mRNA interferase, which may play a role in bacterial resistance to antibiotics. The effect may be overcome by expression of antitoxin HicB.
Characterization
Having showed that the induction of BBa_K3198000 results in growth arrest and suppressed protein production while BBa_K3198001 expression restores growth and protein production, 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 long period of time to still be able to produce sufficient amount of protein.
To test this hypothesis, composite part BBa_K3198007 containing HicA toxin (BBa_K3198000) and LuxABCDE gene (BBa_K325909) is constructed. In this plasmid, BBa_K3198007 is placed under a IPTG-inducible promoter while BBa_K325909 is placed under an arabinose-inducible promoter. Coupled with another composite part (BBa_K3198008) containing HicB antitoxin also under the control of arabinose-inducible promoter, 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_K3198000 originated from Escherichia coli K-12 K12 and its sequence was synthesized by IDT.
Design Considerations
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 549
Illegal NheI site found at 1767
Illegal NheI site found at 4733 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 3731
Illegal BamHI site found at 489
Illegal BamHI site found at 1706
Illegal XhoI site found at 4561 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 1541
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 6120
Illegal BsaI.rc site found at 3129
Illegal SapI site found at 1523
Illegal SapI.rc site found at 6445
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