Difference between revisions of "Part:BBa K4156101"
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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. 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.
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==Improvement==
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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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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===
 
===Characterization===

Revision as of 13:01, 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. 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

lactate induced promoter testing

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.

control
Figure 1: Induction of downstream gene mRFP expression over time by an R reporter consisting of plldR +mRFP in different at different lactate concentrations.


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]