Difference between revisions of "Part:BBa K3198003"
 
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<partinfo>BBa_K3198003 short</partinfo>
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<!-- This part contains the toxin component of a type II toxin-antitoxin (TA) system. HicA is a probable translation-independent mRNA interferase.<br><br> -->
 
 
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K3198002 SequenceAndFeatures</partinfo>
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<partinfo>BBa_K3198003 SequenceAndFeatures</partinfo>
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===Description===
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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===
 
===Usage===
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It is demonstrated that researchers that the RES-Xre locus from </i>Photorhabdus luminescens</i> and other bacterial species function as bona fide TA modules in <i>Escherichia coli</i>.  
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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 <i>Escherichia coli</i> - more specifically, to overcome the pre-induced dormant state of these cells.
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The activation of the toxin in vivo causes a depletion of intracellular NAD+ levels, eventually leading to inhibition of cell growth in <i>E. coli</i> and inhibition of global macromolecular biosynthesis.  
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===Biology===
 
===Biology===
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This part is from the res-xre locus from Photorhabdus luminescens and other bacterial species.
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This part originated from the RES-Xre locus from <i>Photorhabdus luminescens</i> and other bacterial species.  
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===Characterization===
 
===Characterization===
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<!-- Team NUS 2019 has added a new biobrick (BBa_K3198000) into the iGEM repository this year. This biobrick was found to possess a bacteriostatic effect as reported by Gerdes et al in 2008 (doi:10.1128/JB.01013-08) and was therefore used by Team NUS 2019 as part of their sleep-wake module to control the growth of E. coli in their project.
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Team NUS Singapore 2019 hypothesized that the induction of BBa_K3198003 expression will abolish the effect of growth arrest in <i>Escherichia coli</i> and result in resumption of growth comparable to cells not treated with toxin.
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<br><br>(BBa_K3198000) was placed under an IPTG-inducible promoter and various IPTG concentrations were utilized to determine their effect on the growth of native <i>MG1655</i>. In the same plasmid, another cassette containing GFP reporter gene under constitutive promoter was present to enable characterization of the effect of HicA on protein production. 
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<br><br>Characterization of cells transformed with this plasmid was performed at 37°C for 12h continuously. The results showed that IPTG concentrations of 100μM, 500μM and 2mM resulted in growth arrest as shown by a reduction and plateau in OD600. IPTG concentrations beyond 500μM did not show further reduction in OD600.
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<br><br>Figure 1: Growth curve of control <i>MG1655</i> unaffected by IPTG
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<br><br>Figure 2: Growth curve of <i>MG1655</i> transformed with HicA-containing plasmid
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<br><br>Furthermore, team NUS 2019 also studied the effect of (BBa_K3198000) on protein expression. The results demonstrated that HicA-containing cells when induced with IPTG resulted in a drop in total GFP level, as opposed to uninduced HicA-containing cells. This suggests that the effect of (BBa_K3198000) on cell growth is likely to affect its protein expression ability.
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<br><br>Figure 3: Total GFP curve of HicA-plasmid containing cells with different IPTG induction (0M, 100μM, 500μM and 2mM)
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<br><br>In summary, we believe that (BBa_K3198000) is a new BioBrick capable of causing cell growth arrest and suppressing protein expression. -->
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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. <i>Escherichia coli</i> MG1655 was used in this characterization and co-transformed with both plasmids.
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<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 OD<sub>600</sub> 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 OD<sub>600</sub> 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.
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<br><i>Figure 1: MG1655 induced with 2mM IPTG at 1h demonstrate growth arrest at approximately 2.5h.</i>
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<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.
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<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.
  
 
===References===
 
===References===
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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.
 
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.
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===Source===
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BBa_K3198003 was generously provided by Professor Ditlev Brodersen from Aarhus University.
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===Design Considerations===
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Latest revision as of 07:01, 21 October 2019

Xre




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
    COMPATIBLE 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