Part:BBa_K3198003
Xre
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Description
This part contains the antitoxin component of a type II toxin-antitoxin (TA) system. It is shown to counteract RES activity by binding to the toxin counterpart and neutralizing the toxin activity. 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_K3198003) into the iGEM repository this year. This biobrick was found to possess the ability to neutralize the effect of BBa_K3198002 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 - more specifically, to overcome the pre-induced dormant state of these cells.
Biology
This part originated from the RES-Xre locus from Photorhabdus luminescens and other bacterial species.
Characterization
Team NUS Singapore 2019 hypothesized that the induction of BBa_K3198003 expression will abolish the effect of growth arrest in Escherichia coli and result in resumption of growth comparable to cells not treated with toxin.
To test this hypothesis, its toxin counterpart (BBa_K3198002) was placed under an IPTG-inducible promoter while BBa_K3198002 was placed under arabinose-inducible promoter in separate plasmids. Escherichia coli MG1655 was used in this characterization and co-transformed with both plasmids.
Characterization of cells transformed with this plasmid was performed using microplate reader at 37°C for 20h continuously. The cells started off with an OD600 of 0.17 at 0h before they were treated with 2mM IPTG to induce the expression of BBa_K3198002 at 1h. At 3.5h, some cells were induced with arabinose ranging from 0.125%, 0.2% and 0.4% to trigger the induction of BBa_K3198003. The results showed that cells induced with arabinose at 3.5h sees an immediate growth resumption in a dose-dependent manner - with the highest arabinose concentration (0.4%) demonstrating an OD600 comparable to uninduced cells (Figure 1). On the other hand, cells induced with IPTG only demonstrates a delayed growth resumption, probably due to the short half life of toxin BBa_K3198002.
Figure 1: MG1655 induced with 2mM IPTG at 1h demonstrate growth arrest at approximately 2.5h.
Additionally, three of the MG1655 strains treated with 0.125%, 0.2% and 0.4% arabinose at 3.5h demonstrate immediate growth resumption.
Taken together, team NUS Singapore 2019 show that BBa_K3198002 is capable of neutralizing its toxin counterpart effect and result in growth resumption comparable to uninduced cells, in a dose-dependent manner.
References
Skjerning, R. B., Senissar, M., Winther, K. S., Gerdes, K., & Brodersen, D. E. (2018). The RES domain toxins of RES-Xre toxin-antitoxin modules induce cell stasis by degrading NAD . Molecular Microbiology, 111(1), 221–236. doi: 10.1111/mmi.14150
Milunovic, B., diCenzo, G.C., Morton, R.A.and Finan, T.M. (2014) Cell growth inhibition upon deletion of four toxin‐antitoxin loci from the megaplasmids of Sinorhizobium meliloti. Journal of Bacteriology, 196, 811–824.
Source
BBa_K3198003 was generously provided by Professor Ditlev Brodersen from Aarhus University.
Design Considerations
| None |