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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<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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<li>HicA is from the hicAB locus of Escherichia coli K-12.</li>
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</ul><br>
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===Usage===
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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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===Biology===
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HicA is from the hicAB locus of Escherichia coli K-12.
  
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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 MG1655 unaffected by IPTG
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<br><br>Figure 2: Growth curve of MG1655 transformed with HicA-containing plasmid
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<br><br>Furthermore, team NUS 2019 also studied the effect of (HicA ID) 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 (HicA ID) 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 (HicA ID) is a new BioBrick capable of causing cell growth arrest and suppressing protein expression.
 
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===Functional Parameters===
 
===Functional Parameters===
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Revision as of 09:29, 31 August 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]

Usage

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.


Biology

HicA is from the hicAB locus of Escherichia coli K-12.


Characterisation

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.

(BBa_K3198000) was placed under an IPTG-inducible promoter and various IPTG concentrations were utilized to determine their effect on the growth of native MG1655. 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.

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.

Figure 1: Growth curve of control MG1655 unaffected by IPTG

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

Furthermore, team NUS 2019 also studied the effect of (HicA ID) 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 (HicA ID) on cell growth is likely to affect its protein expression ability.

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

In summary, we believe that (HicA ID) is a new BioBrick capable of causing cell growth arrest and suppressing protein expression.