Difference between revisions of "Part:BBa K4156078"

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===Usage and Biology===
 
===Usage and Biology===
  
It is mainly regulated by the transcriptional activator (FNR). In the absence of oxygen, the FNR binds to the [4Fe-4S]2+ cluster to generate a transcriptionally active homodimer. However, in the presence of oxygen, the [4Fe-4S]2+ cluster is degraded and the FNR dimer dissociates into inactive monomers.
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It is mainly regulated by the transcriptional activator (FNR). In the absence of oxygen, the FNR binds to the [4Fe-4S]2+ cluster to generate a transcriptionally active homodimer.<sup>[1]</sup> However, in the presence of oxygen, the [4Fe-4S]2+ cluster is degraded and the FNR dimer dissociates into inactive monomers.<sup>[2]</sup>
  
 
===Characterization===
 
===Characterization===
  
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==Initial Testing of hypoxia Promoter==
  
==hypoxia induced promoter testing==
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To Characterize part,we first added mRFP after the promoter and wanted to initially test the response of this promoter to anoxia based on the fluorescence intensity. E. coli Nissle 1917 was used as chassis.Details of the characterization and test results can be found at <html><a style="padding: 0px; margin: 0px;" href="https://parts.igem.org/Part:BBa_K4156110"> BBa_K4156110 </a></html>
  
We constructed a hypoxia reporter consisting of the hypoxia-inducible promoter pPepT+mRFP. To test its performance, we added reporter in different chassis organisms.Fig 1,2 indicates that pPepT induces the expression of the downstream gene mRFP with the decrease of O2. Thus, it can be seen that the hypoxia reporter can work properly.
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==Stability improvement==
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Then,amplifying genetic switches and Boolean logic gates based on serine integrase (TP901) are used in the design of biosensor systems <sup>[3]</sup>. These genetic devices enable bacteria to perform reliable detection, multiplex logic and data storage of clinical biomarkers in human clinical samples <sup>[4-5]</sup> to meet the requirements of medical testing. For characterization, we added switch, which is TP901 and XOR gate, then followed with mRFP. Details of the characterization and test results can be found at <html><a style="padding: 0px; margin: 0px;" href="https://parts.igem.org/Part:BBa_K4156108"> BBa_K4156108 </a></html>
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==Addition of lysis genes==
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Because we have therapeutic proteins that cannot be exocytosed, it is not enough to simply stabilize the response signal, and we intend to add bacteriophage lysis gene phiX174E parts that will enable bacteria lysis.So next we added phiX174E to the above genetic parts. Details of the characterization and test results can be found at <html><a style="padding: 0px; margin: 0px;" href="https://parts.igem.org/Part:BBa_K4156109"> BBa_K4156109 </a></html>
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==Better Chassis==
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Finally, based on the above validation, we can assume that strains were constructed that can stably respond to anoxia. Since the chassis organism must be E. coli, but we started to think in which strain this gene circuit is responding better. So we compared it in E. coli Nissle 1917 and E. coli DH 5-alpha. The data were recorded at 2-hour intervals over 48 hours of induction at the same anoxia condition as before, and finally plotted as the normalized fluorescence intensity (figure 1). It can be observed that the circuit responds with higher intensity in E. coli Nissle 1917 than in E. coli DH5-alpha, so E. coli Nissle 1917 is a better chassis organism.
  
 
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<figure style="text-align:center;">
 
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                 <img style="max-width:700px;" src="https://static.igem.wiki/teams/4156/wiki/part/2-1.png" alt="control">
 
                 <img style="max-width:700px;" src="https://static.igem.wiki/teams/4156/wiki/part/2-1.png" alt="control">
                 <figcaption><b>Figure 1:</b> Induction of downstream gene mRFP expression under hypoxic and normoxic conditions in different chassis organisms over 48h.</figcaption>
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                 <figcaption><b>Figure 1:</b> Induction of downstream gene mRFP expression with different pH values in different chassis organisms over 48h.</figcaption>
 
               </figure>
 
               </figure>
 
</html>
 
</html>
  
<html>
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<figure style="text-align:center;">
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===References===
                <img style="max-width:700px;" src="https://static.igem.wiki/teams/4156/wiki/part/2-2-5-2.png" alt="control">
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<i>
                <figcaption><b>Figure 2:</b> Induction of downstream gene mRFP expression over time by a hypoxia reporter consisting of pPepT+mRFP under hypoxic and normoxic conditions.</figcaption>
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1 Yu B, Yang M, Shi L, et al. Explicit hypoxia targeting with tumor suppression by creating an "obligate" anaerobic Salmonella Typhimurium strain. Sci Rep. 2012;2:436. doi:10.1038/srep00436
              </figure>
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</html>
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2 Goers L, Ainsworth C, Goey CH, Kontoravdi C, Freemont PS, Polizzi KM. Whole-cell Escherichia coli lactate biosensor for monitoring mammalian cell cultures during biopharmaceutical production. Biotechnol Bioeng. 2017;114(6):1290-1300. doi:10.1002/bit.26254
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3 Courbet A, Endy D, Renard E, Molina F, Bonnet J. Detection of pathological biomarkers in human clinical samples via amplifying genetic switches and logic gates. Sci Transl Med. May 27 2015;7(289):289ra83. doi:10.1126/scitranslmed.aaa3601
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4 Benenson Y. Biomolecular computing systems: principles, progress and potential. Nat Rev Genet. Jun 12 2012;13(7):455-68. doi:10.1038/nrg3197
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5 Bonnet J, Yin P, Ortiz ME, Subsoontorn P, Endy D. Amplifying genetic logic gates. Science. May 3 2013;340(6132):599-603. doi:10.1126/science.1232758
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</i>
  
 
<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>

Revision as of 08:17, 11 October 2022


-- No description --

pPepT is a hypoxia-sensing promoter, designed to sense oxygen.


Usage and Biology

It is mainly regulated by the transcriptional activator (FNR). In the absence of oxygen, the FNR binds to the [4Fe-4S]2+ cluster to generate a transcriptionally active homodimer.[1] However, in the presence of oxygen, the [4Fe-4S]2+ cluster is degraded and the FNR dimer dissociates into inactive monomers.[2]

Characterization

Initial Testing of hypoxia Promoter

To Characterize part,we first added mRFP after the promoter and wanted to initially test the response of this promoter to anoxia based on the fluorescence intensity. E. coli Nissle 1917 was used as chassis.Details of the characterization and test results can be found at BBa_K4156110

Stability improvement

Then,amplifying genetic switches and Boolean logic gates based on serine integrase (TP901) are used in the design of biosensor systems [3]. These genetic devices enable bacteria to perform reliable detection, multiplex logic and data storage of clinical biomarkers in human clinical samples [4-5] to meet the requirements of medical testing. For characterization, we added switch, which is TP901 and XOR gate, then followed with mRFP. Details of the characterization and test results can be found at BBa_K4156108


Addition of lysis genes

Because we have therapeutic proteins that cannot be exocytosed, it is not enough to simply stabilize the response signal, and we intend to add bacteriophage lysis gene phiX174E parts that will enable bacteria lysis.So next we added phiX174E to the above genetic parts. Details of the characterization and test results can be found at BBa_K4156109


Better Chassis

Finally, based on the above validation, we can assume that strains were constructed that can stably respond to anoxia. Since the chassis organism must be E. coli, but we started to think in which strain this gene circuit is responding better. So we compared it in E. coli Nissle 1917 and E. coli DH 5-alpha. The data were recorded at 2-hour intervals over 48 hours of induction at the same anoxia condition as before, and finally plotted as the normalized fluorescence intensity (figure 1). It can be observed that the circuit responds with higher intensity in E. coli Nissle 1917 than in E. coli DH5-alpha, so E. coli Nissle 1917 is a better chassis organism.

control
Figure 1: Induction of downstream gene mRFP expression with different pH values in different chassis organisms over 48h.


References

1 Yu B, Yang M, Shi L, et al. Explicit hypoxia targeting with tumor suppression by creating an "obligate" anaerobic Salmonella Typhimurium strain. Sci Rep. 2012;2:436. doi:10.1038/srep00436

2 Goers L, Ainsworth C, Goey CH, Kontoravdi C, Freemont PS, Polizzi KM. Whole-cell Escherichia coli lactate biosensor for monitoring mammalian cell cultures during biopharmaceutical production. Biotechnol Bioeng. 2017;114(6):1290-1300. doi:10.1002/bit.26254

3 Courbet A, Endy D, Renard E, Molina F, Bonnet J. Detection of pathological biomarkers in human clinical samples via amplifying genetic switches and logic gates. Sci Transl Med. May 27 2015;7(289):289ra83. doi:10.1126/scitranslmed.aaa3601

4 Benenson Y. Biomolecular computing systems: principles, progress and potential. Nat Rev Genet. Jun 12 2012;13(7):455-68. doi:10.1038/nrg3197

5 Bonnet J, Yin P, Ortiz ME, Subsoontorn P, Endy D. Amplifying genetic logic gates. Science. May 3 2013;340(6132):599-603. doi:10.1126/science.1232758

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]