Difference between revisions of "Part:BBa K3198002"
 
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<partinfo>BBa_K3198002 short</partinfo>
 
<partinfo>BBa_K3198002 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>
 
<partinfo>BBa_K3198002 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K3198002 SequenceAndFeatures</partinfo>
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===Description===
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This part contains the toxin component of a type II toxin-antitoxin (TA) system. RES is orthologous to <i>Escherichia coli</i> and was named after three conserved amino acids, arginine (R), glutamate (E) and serine (S). 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 Photorhabdus luminescens and other bacterial species function as bona fide TA modules in Escherichia coli.  
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Team NUS Singapore 2019 has added a new biobrick (BBa_K3198002) into the iGEM repository this year. This biobrick was found to possess the ability to inhibit cell growth by acting as a NAD+ glycohydrolase (NADase). 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> by inducing metabolite-mediated dormancy in 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 E. coli 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. The activation of this toxin in vivo has been shown to deplete intracellular NAD+ levels, subsequently resulting in the inhibition of global macromolecular biosynthesis as well as cell growth in <i>Escherichia coli</i>.
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===Characterisation===
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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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<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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===Characterization===
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Team NUS Singapore 2019 hypothesized that the induction of BBa_K3198002 expression would have resulted in reduced growth, supported by reduced OD<sub>600</sub> which is an indicator of biomass.
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<br><br>
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To test this hypothesis, BBa_K3198002 which was originally provided by Professor Brodersen was placed under an arabinose-inducible promoter. Arabinose concentrations of 0.2% and 0.4% were explored to determine their effect on the growth of native <i>Escherichia coli</i> MG1655.
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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 0.1 at 0h before they were treated with both 0.2% and 0.4% arabinose at 1h to induce the expression of BBa_K3198002.
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<br><br>
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The team observed that the OD<sub>600</sub> of the cells treated with arabinose plateaued at approximately 2.5h, indicating cell growth arrest (Figure 1). Interestingly, both cells treated with 0.2% and 0.4% arabinose demonstrated a resumption in growth at about 12.5h. It is unlikely to be that of BBa_K3198002’s cognate antitoxin, since the transformed plasmid does not contain any antitoxin gene and RES/Xre is orthologous to <i>Escherichia coli</i>. As such, the team hypothesized that either arabinose has a positive effect on cell growth, or that BBa_K3198002 has a relatively short half life and it degrades after approximately 11.5h to result in growth resumption.
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<img style="width:400px" src="https://2019.igem.org/wiki/images/a/ad/T--NUS_Singapore--PartsRegistry_RES1.png">
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<br><i>Figure 1: Professor Brodersen’s MG1655 containing RES plasmid treated with 0.2% and 0.4% arabinose respectively at 1h.</i>
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<br><br>To test this hypothesis, BBa_K3198002 was cloned under an IPTG-inducible promoter and 2mM IPTG concentration was explored to determine their effect on the growth of native MG1655, using the same characterization conditions and set-up.
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<br><br>Similarly, the cells induced with 2mM IPTG showed a plateau in OD<sub>600</sub> almost immediately after induction (Figure 2). However, the automatic resumption in growth remained consistent even when BBa_K3198002 was cloned under an IPTG-inducible promoter. It is also noteworthy that the resumption was earlier in this genetic circuit as compared to Professor Brodersen’s original plasmid.
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<img style="width:400px" src="https://2019.igem.org/wiki/images/4/44/T--NUS_Singapore--PartsRegistry_RES2.png">
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<br><i>Figure 2: MG1655 containing RES and Xre plasmids treated with 2mM IPTG at 1h.</i>
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<br><br>Taken together, these results validated BBa_K3198002 ability to inhibit cell growth, but for a relatively short period of time when controlled by both arabinose- and IPTG-inducible systems.
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===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_K3198002 was generously provided by Professor Ditlev Brodersen from Aarhus University.
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===Design Considerations===
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Latest revision as of 07:00, 21 October 2019

RES




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 toxin component of a type II toxin-antitoxin (TA) system. RES is orthologous to Escherichia coli and was named after three conserved amino acids, arginine (R), glutamate (E) and serine (S). 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_K3198002) into the iGEM repository this year. This biobrick was found to possess the ability to inhibit cell growth by acting as a NAD+ glycohydrolase (NADase). For this reason, team NUS Singapore 2019 used this biobrick as part of their sleep-wake module to control the growth of Escherichia coli by inducing metabolite-mediated dormancy in these cells.

Biology

This part originated from the RES-Xre locus from Photorhabdus luminescens and other bacterial species. The activation of this toxin in vivo has been shown to deplete intracellular NAD+ levels, subsequently resulting in the inhibition of global macromolecular biosynthesis as well as cell growth in Escherichia coli.

Characterization

Team NUS Singapore 2019 hypothesized that the induction of BBa_K3198002 expression would have resulted in reduced growth, supported by reduced OD600 which is an indicator of biomass.

To test this hypothesis, BBa_K3198002 which was originally provided by Professor Brodersen was placed under an arabinose-inducible promoter. Arabinose concentrations of 0.2% and 0.4% were explored to determine their effect on the growth of native Escherichia coli MG1655.

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 0.1 at 0h before they were treated with both 0.2% and 0.4% arabinose at 1h to induce the expression of BBa_K3198002.

The team observed that the OD600 of the cells treated with arabinose plateaued at approximately 2.5h, indicating cell growth arrest (Figure 1). Interestingly, both cells treated with 0.2% and 0.4% arabinose demonstrated a resumption in growth at about 12.5h. It is unlikely to be that of BBa_K3198002’s cognate antitoxin, since the transformed plasmid does not contain any antitoxin gene and RES/Xre is orthologous to Escherichia coli. As such, the team hypothesized that either arabinose has a positive effect on cell growth, or that BBa_K3198002 has a relatively short half life and it degrades after approximately 11.5h to result in growth resumption.
Figure 1: Professor Brodersen’s MG1655 containing RES plasmid treated with 0.2% and 0.4% arabinose respectively at 1h.

To test this hypothesis, BBa_K3198002 was cloned under an IPTG-inducible promoter and 2mM IPTG concentration was explored to determine their effect on the growth of native MG1655, using the same characterization conditions and set-up.

Similarly, the cells induced with 2mM IPTG showed a plateau in OD600 almost immediately after induction (Figure 2). However, the automatic resumption in growth remained consistent even when BBa_K3198002 was cloned under an IPTG-inducible promoter. It is also noteworthy that the resumption was earlier in this genetic circuit as compared to Professor Brodersen’s original plasmid.


Figure 2: MG1655 containing RES and Xre plasmids treated with 2mM IPTG at 1h.

Taken together, these results validated BBa_K3198002 ability to inhibit cell growth, but for a relatively short period of time when controlled by both arabinose- and IPTG-inducible systems.

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_K3198002 was generously provided by Professor Ditlev Brodersen from Aarhus University.

Design Considerations