Difference between revisions of "Part:BBa K4156101"
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===Usage and Biology===
 
===Usage and Biology===
  
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Llprd operon is composed of the lldR regulatory protein (<html><a style="padding: 0px; margin: 0px;" href="https://parts.igem.org/Part:BBa_K1847001"> BBa_K1847001 </a></html>), the terminator B0012, and the LldPRD promoter（<html><a style="padding: 0px; margin: 0px;" href="https://parts.igem.org/Part:BBa_M36021"> BBa_M36021 </a></html>）. At the LldPRD promoter, there are two operator sites O1 and O2 , which are able to bind to the lldR protein and block subsequent gene transcription. The presence of lactic acid molecules can unblock this blocking phenomenon. Thus, the Llprd operon can specifically respond to lactate and activate downstream genes. In our experiments, we used Llprd operon to adapt to the high lactate characteristics of the tumor cell microenvironment. Enabling our therapeutic strains to rapidly target to tumor cells and achieve targeted therapy.
+
Llprd operon is composed of the lldR regulatory protein (<html><a style="padding: 0px; margin: 0px;" href="https://parts.igem.org/Part:BBa_K1847001"> BBa_K1847001 </a></html>), the terminator B0012, and the LldPRD promoter（<html><a style="padding: 0px; margin: 0px;" href="https://parts.igem.org/Part:BBa_M36021"> BBa_M36021 </a></html>）. At the LldPRD promoter, there are two operator sites O1 and O2 , which are able to bind to the lldR protein and block subsequent gene transcription. The presence of lactic acid molecules can unblock this blocking phenomenon.<sup>[1-2]</sup> Thus, the Llprd operon can specifically respond to lactate and activate downstream genes. In our experiments, we used Llprd operon to adapt to the high lactate characteristics of the tumor cell microenvironment. Enabling our therapeutic strains to rapidly target to tumor cells and achieve targeted therapy.
 
==Improvement==
 
==Improvement==
 +
 
We built on the previously created parts lldPRD operon promoter（<html><a style="padding: 0px; margin: 0px;" href="https://parts.igem.org/Part:BBa_M36021"> BBa_M36021 </a></html>) and lldR ( <html><a style="padding: 0px; margin: 0px;" href="https://parts.igem.org/Part:BBa_K1847001"> BBa_K1847001 </a></html>) by introducing a combination of hyper-spank and RBS spoVG in its head and an optimized sequence of RiboJ in its tail, once created a new LlprD operon. Most importantly, we combined the constructed LlprD operon with the TP901-mediated XOR logic gate, which greatly improved the performance of the LlprD operon.  
 
We built on the previously created parts lldPRD operon promoter（<html><a style="padding: 0px; margin: 0px;" href="https://parts.igem.org/Part:BBa_M36021"> BBa_M36021 </a></html>) and lldR ( <html><a style="padding: 0px; margin: 0px;" href="https://parts.igem.org/Part:BBa_K1847001"> BBa_K1847001 </a></html>) by introducing a combination of hyper-spank and RBS spoVG in its head and an optimized sequence of RiboJ in its tail, once created a new LlprD operon. Most importantly, we combined the constructed LlprD operon with the TP901-mediated XOR logic gate, which greatly improved the performance of the LlprD operon.  
  
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===Characterization===
 
===Characterization===
  
 +
==Initial Testing of lactate Promoter==
 +
 +
To Characterize part，we first added mRFP after the promoter and wanted to initially test the response of this promoter to different lactate concerntartions 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_K4156114"> BBa_K4156114 </a></html>
 +
 +
==Stability improvement==
 +
 +
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_K4156104"> BBa_K4156104 </a></html>
 +
 +
 +
==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 <html><a style="padding: 0px; margin: 0px;" href="https://parts.igem.org/Part:BBa_K4156105"> BBa_K4156105 </a></html>
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 +
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===References===
 +
<i>
 +
1 Aguilera L, Campos E, Giménez R, Badía J, Aguilar J, Baldoma L. Dual role of LldR in regulation of the lldPRD operon, involved in L-lactate metabolism in Escherichia coli. J Bacteriol. Apr 2008;190(8):2997-3005. doi:10.1128/jb.02013-07
 +
 +
2 Weghoff MC, Bertsch J, Müller V. A novel mode of lactate metabolism in strictly anaerobic bacteria. Environ Microbiol. Mar 2015;17(3):670-7. doi:10.1111/1462-2920.12493
  
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==lactate induced promoter testing==
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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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We linked mRFP downstream of the lactate-sensing promoter to construct an R reporter, and determined the promoter response to lactate by detecting the fluorescence of RFP. In the Fig 1, the homogenized fluorescence intensity (mRFP/Cell) of the lactate (plldR) induced reporters is shown.It indicates that plldR induces the expression of the downstream gene mRFP with the increase of lactate concertration. Thus, it can be seen that the lactater reporter can work properly.
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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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<html>
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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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<figure style="text-align:center;">
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                <img style="max-width:700px;" src="https://static.igem.wiki/teams/4156/wiki/part/3-1-6-2.png" alt="control">
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                <figcaption><b>Figure 1:</b> Induction of downstream gene mRFP expression over time by an R reporter consisting of plldR +mRFP in different at different lactate concentrations.</figcaption>
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              </figure>
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</html>
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</i>
  
 
<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>

Revision as of 13:06, 11 October 2022


pLldR

An Operon unit consisting of a collection of LldR-regulated promoters, and is capable of responding to lactate concentration.


Usage and Biology

Llprd operon is composed of the lldR regulatory protein ( BBa_K1847001 ), the terminator B0012, and the LldPRD promoter（ BBa_M36021 ）. At the LldPRD promoter, there are two operator sites O1 and O2 , which are able to bind to the lldR protein and block subsequent gene transcription. The presence of lactic acid molecules can unblock this blocking phenomenon.[1-2] Thus, the Llprd operon can specifically respond to lactate and activate downstream genes. In our experiments, we used Llprd operon to adapt to the high lactate characteristics of the tumor cell microenvironment. Enabling our therapeutic strains to rapidly target to tumor cells and achieve targeted therapy.

Improvement

We built on the previously created parts lldPRD operon promoter（ BBa_M36021 ) and lldR ( BBa_K1847001 ) by introducing a combination of hyper-spank and RBS spoVG in its head and an optimized sequence of RiboJ in its tail, once created a new LlprD operon. Most importantly, we combined the constructed LlprD operon with the TP901-mediated XOR logic gate, which greatly improved the performance of the LlprD operon.

The optimized LlprD operon improves the signal output level while the signal output level is more stable. This is undoubtedly beneficial for the subsequent construction. More notably, we introduced the TP901-XOR gate system, which helps to coordinate many different inducible operons.We characterized our optimized system using LlprD-mRFP as a control as follows.

Characterization

Initial Testing of lactate Promoter

To Characterize part，we first added mRFP after the promoter and wanted to initially test the response of this promoter to different lactate concerntartions 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_K4156114

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_K4156104


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_K4156105


References

1 Aguilera L, Campos E, Giménez R, Badía J, Aguilar J, Baldoma L. Dual role of LldR in regulation of the lldPRD operon, involved in L-lactate metabolism in Escherichia coli. J Bacteriol. Apr 2008;190(8):2997-3005. doi:10.1128/jb.02013-07

2 Weghoff MC, Bertsch J, Müller V. A novel mode of lactate metabolism in strictly anaerobic bacteria. Environ Microbiol. Mar 2015;17(3):670-7. doi:10.1111/1462-2920.12493

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
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 1
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 688
  • 1000
    COMPATIBLE WITH RFC[1000]