Difference between revisions of "Part:BBa K3198000"
 
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<partinfo>BBa_K3198001 short</partinfo>
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<partinfo>BBa_K3198000 short</partinfo>
  
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This part contains the antitoxin component of a type II toxin-antitoxin (TA) system. It functions as an mRNA interferase antitoxin; overexpression prevents HicA-mediated cessation of cell growth and cell death.
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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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HicB functions as an mRNA interferase antitoxin. Expression of HicB (145 aa) prevented HicA-mediated inhibition of cell growth. HicB neutralizes HicA and therefore functions as an antitoxin. HicB could resuscitate cells inhibited by HicA.<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_K3198001 SequenceAndFeatures</partinfo>
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<partinfo>BBa_K3198000 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. HicA is a probable translation-independent mRNA interferase. Refer to our wiki https://2019.igem.org/Team:NUS_Singapore/Design#Characterization for more details.
  
 
===Usage===
 
===Usage===
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HicB functions as an mRNA interferase antitoxin. Expression of HicB (145 aa) prevented HicA-mediated inhibition of cell growth. HicB neutralizes HicA and therefore functions as an antitoxin. HicB could resuscitate cells inhibited by HicA.
+
Team NUS Singapore 2019 has added a new biobrick (BBa_K3198000) 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 <i>Escherichia coli</i> by inducing dormancy in these cells.
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===Biology===
 
===Biology===
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HicB is from hicAB locus of Escherichia coli K-12.  
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HicA originates from the hicAB locus of <i>Escherichia coli</i> 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. In addition, overexpression of HicA leads to cell death and inhibits cell proliferation via inhibition of translation. The effect may be overcome by expression of antitoxin HicB.
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===Characterization===
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Team NUS Singapore 2019 hypothesized that the induction of BBa_K3198000 will result in reduced growth, supported by reduced OD<sub>600</sub> which is an indicator of biomass. With the growth arrested, the team also hypothesized that there will be a reduction in protein production since the mechanism of this biobrick targets global translation to cause growth arrest.
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<br><br>To test this hypothesis, BBa_K3198000 was placed under an IPTG-inducible promoter and various IPTG concentrations were explored to determine their effect on the growth of native MG1655. In the same plasmid, another cassette containing a GFP reporter gene under constitutive promoter was present to enable the characterization of HicA effect on protein production.
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<br><br>Characterization of cells transformed with this plasmid was performed using a microplate reader 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 OD<sub>600</sub> (Figure 2). IPTG concentrations beyond 500μM did not show further reduction in OD<sub>600</sub>.
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<br><br>
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<html>
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<img style="width:400px" src="https://2019.igem.org/wiki/images/2/24/T--NUS_Singapore--PartsRegistry_HicA1.png">
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</html>
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<br><i>Figure 1: Growth curve of control MG1655 unaffected by IPTG</i>
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<br><br>
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<html>
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<img style="width:400px" src="https://2019.igem.org/wiki/images/1/15/T--NUS_Singapore--PartsRegistry_HicA2.png">
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</html>
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<br><i>Figure 2: Growth curve of MG1655 transformed with HicA-containing plasmid</i>
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<br><br>Furthermore, team NUS Singapore 2019 also studied the effect of BBa_K3198000 on protein production. 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 (Figure 3). This suggests that the effect of BBa_K3198000 on cell growth is likely to also affect protein production in these cells.
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<br><br>
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<html>
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<img style="width:400px" src="https://2019.igem.org/wiki/images/0/06/T--NUS_Singapore--PartsRegistry_HicA3.png">
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</html>
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<br><i>Figure 3: Total GFP curve of HicA-plasmid containing cells with different IPTG induction (0M, 100μM, 500μM and 2mM)</i>
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<br><br>Moving forward, the team hypothesized that there exists a certain IPTG concentration threshold which could serve as minimum IPTG concentration to see an effect on cell growth (Figure 4). As such, the team explored a lower range of IPTG concentrations ranging from 10μM to 500μM using the same characterization conditions. Indeed, the results demonstrate that IPTG concentration of 10μM has no effect on cell growth. Instead, the minimum IPTG concentration which demonstrate growth reduction was 25μM.
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<br><br>
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<html>
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<img style="width:400px" src="https://2019.igem.org/wiki/images/a/ac/T--NUS_Singapore--PartsRegistry_HicA4.png">
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</html>
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<br><i>Figure 4: Growth curve of MG1655 transformed with HicA-containing plasmid at a lower range of IPTG concentrations</i>
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<br><br>In summary, team NUS Singapore 2019 believes that BBa_K3198000 is a new BioBrick capable of causing cell growth arrest and suppressing protein expression, more specifically when a minimum IPTG concentration of 25μM was used.
  
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===References===
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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.
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<br><br>
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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
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<br><br>
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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
  
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===Characterisation===
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===Sources===
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Team NUS 2019 has added another new biobrick (BBa_K3198001) into iGEM repository this year. This biobrick was found to possess the ability to neutralize the effect of HicA( biobrick id) and therefore functions as an antitoxin. For this reason, team NUS 2019 used this biobrick as part of their sleep-wake module to control the growth of E. coli in their project.
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BBa_K3198000 originated from <i>Escherichia coli</i> K12 and its sequence was synthesized by IDT.
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<br><br>(BBa_K3198001) was placed under an arabinose-inducible promoter and various arabinose concentrations were used to determine their ability to resume cell growth native MG1655.
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<br><br>Characterization of cells transformed with this plasmid was performed at 37°C for 12h continuously. Both toxin and antitoxin inducers were added together at 0h, with IPTG concentration fixed at 500uM with varying arabinose concentrations of 0M, 0.0266M, 0.133M and 0.266M.
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<br><br>After the addition of 500μM IPTG into cells co-transformed with (BBa_K3198000) and (BBa_K3198001) plasmid, it was shown that even at the lowest arabinose concentration of 0.0266M, the growth of the cells was able to restore to the same level as uninduced cells. On the other hand, cells only induced with 500μM IPTG did not managed to show growth resumption in the absence of arabinose. This demonstrated the function of (BBa_K3198001) as an antitoxin capable of neutralising (BBa_K3198000) effect on native MG1655 growth.  
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===Design Considerations===
  
 
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Latest revision as of 06:59, 21 October 2019

HicA

This part contains the toxin component of a type II toxin-antitoxin (TA) system. HicA is a probable translation-independent mRNA interferase.

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. HicA is a probable translation-independent mRNA interferase. 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_K3198000) 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 by inducing dormancy 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. In addition, overexpression of HicA leads to cell death and inhibits cell proliferation via inhibition of translation. The effect may be overcome by expression of antitoxin HicB.

Characterization

Team NUS Singapore 2019 hypothesized that the induction of BBa_K3198000 will result in reduced growth, supported by reduced OD600 which is an indicator of biomass. With the growth arrested, the team also hypothesized that there will be a reduction in protein production since the mechanism of this biobrick targets global translation to cause growth arrest.

To test this hypothesis, BBa_K3198000 was placed under an IPTG-inducible promoter and various IPTG concentrations were explored to determine their effect on the growth of native MG1655. In the same plasmid, another cassette containing a GFP reporter gene under constitutive promoter was present to enable the characterization of HicA effect on protein production.

Characterization of cells transformed with this plasmid was performed using a microplate reader 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 (Figure 2). IPTG concentrations beyond 500μM did not show further reduction in OD600.


Figure 1: Growth curve of control MG1655 unaffected by IPTG


Figure 2: Growth curve of MG1655 transformed with HicA-containing plasmid

Furthermore, team NUS Singapore 2019 also studied the effect of BBa_K3198000 on protein production. 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 (Figure 3). This suggests that the effect of BBa_K3198000 on cell growth is likely to also affect protein production in these cells.


Figure 3: Total GFP curve of HicA-plasmid containing cells with different IPTG induction (0M, 100μM, 500μM and 2mM)

Moving forward, the team hypothesized that there exists a certain IPTG concentration threshold which could serve as minimum IPTG concentration to see an effect on cell growth (Figure 4). As such, the team explored a lower range of IPTG concentrations ranging from 10μM to 500μM using the same characterization conditions. Indeed, the results demonstrate that IPTG concentration of 10μM has no effect on cell growth. Instead, the minimum IPTG concentration which demonstrate growth reduction was 25μM.


Figure 4: Growth curve of MG1655 transformed with HicA-containing plasmid at a lower range of IPTG concentrations

In summary, team NUS Singapore 2019 believes that BBa_K3198000 is a new BioBrick capable of causing cell growth arrest and suppressing protein expression, more specifically when a minimum IPTG concentration of 25μM was used.

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

Sources

BBa_K3198000 originated from Escherichia coli K12 and its sequence was synthesized by IDT.

Design Considerations