Difference between revisions of "Part:BBa K3198000"
 
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<partinfo>BBa_K3198000 short</partinfo><br><br>
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<partinfo>BBa_K3198000 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.
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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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<!-- Add more about the biology of this part here
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<span class='h3bb'>Sequence and Features</span>
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===Usage and Biology===
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<partinfo>BBa_K3198000 SequenceAndFeatures</partinfo>
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<ul>
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<li>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.</li>
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===Description===
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<li>HicA is from the hicAB locus of Escherichia coli K-12.</li>
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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.
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</ul><br>
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===Usage===
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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===
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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.
  
 
===Characterization===
 
===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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<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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<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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<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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<b>Summary of Results as in https://parts.igem.org/Part:BBa_K2819118</b>
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===References===
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<ul>
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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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  <li>PhtpG1-mRFP <u><b>can work in different genetic backgrounds</b></u>. Interestingly, the stress reporter was robust in all three strains of <i>E. coli</i> that it was tested in: DH5α, BL21 (DE3) and BL21 Star (DE3).</li>
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<br><br>
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  <li>PhtpG1-mRFP is <b><u>robust in different temperatures</u></b>. However, at low temperatures i.e. 25°C, more time is expected to allow for growth to stabilize/increase steadily before readings follow an expected trend. Given that PhtpG1 is a promoter that is involved in heat-shock response (and that requires heat-shock response sigma factor (σ32)), higher temperatures would be expected to activate the promoter to a greater extent (Ceroni et al., 2018). </li>
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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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  <li>PhtpG1-mRFP appears to be <b><u>sensitive to the size of constructs that is introduced</u></b> into the cell. The highest levels of mRFP was consistently found to be generated in test construct Set-up C (see Figure 6), in which the large de novo plasmid<sup>a</sup> was expressed, followed by test construct Set-up E and F expressing the moderately sized FNS<sup>a</sup> protein, and the lowest being in test construct Set-up A, in which only GFP was expressed<sup>a</sup>.</li>
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<br><br>
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</ul>
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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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<font size="1"><sup>a</sup>Sizes of proteins:  MCS (55 kDa) [de novo plasmid], OsPKS (43 kDa) [de novo plasmid], 4CL (59 kDa) [de novo plasmid], GFP (27 kDa) and FNS (41 kDa).</font><br><br>
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===Sources===
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BBa_K3198000 originated from <i>Escherichia coli</i> K12 and its sequence was synthesized by IDT.
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===Design Considerations===
  
 
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K3198000 SequenceAndFeatures</partinfo>
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===Functional Parameters===
 
===Functional Parameters===
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<partinfo>BBa_K3198000 parameters</partinfo>
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<partinfo>BBa_K3198001 parameters</partinfo>
 
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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