Difference between revisions of "Part:BBa K3198000"
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__NOTOC__
 
__NOTOC__
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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>
  
 
===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.
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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.
  
  
 
===Biology===
 
===Biology===
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HicB is from hicAB locus of Escherichia coli K-12.  
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HicA is from the hicAB locus of Escherichia coli K-12.
  
  
 
===Characterisation===
 
===Characterisation===
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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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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_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>(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. 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>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>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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<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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Revision as of 09:36, 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 (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.

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

In summary, we believe that (BBa_K3198000) is a new BioBrick capable of causing cell growth arrest and suppressing protein expression.