Difference between revisions of "Part:BBa K3198002"
Tiantian0412 (Talk | contribs) |
|||
Line 1: | Line 1: | ||
+ | |||
__NOTOC__ | __NOTOC__ | ||
− | <partinfo> | + | <partinfo>BBa_K3198002 short</partinfo> |
<!-- | <!-- | ||
− | This part contains the | + | 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. |
+ | |||
<span class='h3bb'>Sequence and Features</span> | <span class='h3bb'>Sequence and Features</span> | ||
Line 9: | Line 11: | ||
===Usage=== | ===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 E. coli - more specifically, to overcome the pre-induced dormant state of these cells. | |
− | + | ||
− | + | ||
− | + | ||
− | + | ||
===Biology=== | ===Biology=== | ||
Line 20: | Line 19: | ||
===Characterization=== | ===Characterization=== | ||
− | + | Team NUS Singapore 2019 hypothesized that the induction of BBa_K3198003 expression will abolish the effect of growth arrest in E. coli and result in resumption of growth comparable to cells not treated with toxin. | |
− | <br><br>( | + | <br><br>To test this hypothesis, its toxin counterpart (BBa_K3198003) was placed under an IPTG-inducible promoter while BBa_K3198002 was placed under arabinose-inducible promoter in separate plasmids. E. coli MG1655 was used in this characterization and co-transformed with both plasmids. |
− | <br><br>Characterization of cells transformed with this plasmid was performed at 37°C for | + | <br><br>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_K3198003 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_K3198002. 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. On the other hand, cells induced with IPTG only demonstrates a delayed growth resumption, probably due to the short half life of toxin BBa_K3198003. |
− | <br><br> | + | <br><br>https://2019.igem.org/wiki/images/a/a4/T--NUS_Singapore--PartsRegistry_Xre1.png |
− | <br><br>Figure | + | |
− | + | <br><br>Figure 1: MG1655 induced with 2mM IPTG at 1h demonstrate growth arrest at approximately 2.5h. | |
− | <br><br> | + | <br><br>Additionally, three of the MG1655 strains treated with 0.125%, 0.2% and 0.4% arabinose at 3.5h demonstrate immediate growth resumption. |
− | <br><br> | + | |
+ | <br><br> 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. | ||
+ | |||
+ | |||
Line 34: | Line 36: | ||
<br><br> | <br><br> | ||
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 | 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 | ||
+ | |||
+ | |||
+ | ===Sources=== | ||
+ | BBa_K3198003 was generously provided by Professor Ditlev Brodersen from Aarhus University. | ||
+ | |||
+ | ===Design Considerations=== | ||
<!-- --> | <!-- --> |
Revision as of 08:05, 12 September 2019
RES