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===Characterization===
 
===Characterization===
  
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==lactate induced promoter testing==
  
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==In vitro characterization and data analysis of the reported strains==
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We constructed a lactate  reporter consisting of the lactate-inducible promoter pLldR+mRFP. To test its performance, we added reporter in different chassis organisms.Fig 1 indicates that pLldR induces the expression of the downstream gene mRFP with the decrease of O2. Thus, it can be seen that the lactate reporter can work properly.
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To improve signaling stability as well as accuracy, we added Amplifying genetic switches based on serine integrase (Bxb1,TP901) to the R reporter(<html><a style="padding: 0px; margin: 0px;" href="https://parts.igem.org/Part:BBa_K4156114"> BBa_K4156115 </a></html> ) to construct the AR reporter.
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Fig 1 indicates lactate (plldR) induced AR reporter with homogenized fluorescence intensity (mRFP/Cell). Comparing Fig1, 2, it can be seen that the fluorescence intensity of the AR reporter decreased significantly at a lactate concentration of 0 mM, and its expression was more stable over time. The fluorescence intensity of the AR reporter was also greater at other concentrations of lactate induction, and the difference between the fluorescence intensity after lactate induction at each concentration was more pronounced. This result indicates that the addition of amplifying genetic switch enhances the reporter intensity and robustness of the lactate biosensor.
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                <img style="max-width:700px;" src="https://static.igem.wiki/teams/4156/wiki/part/6-1.png" alt="control">
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                <figcaption><b>Figure 1:</b> Induction of downstream gene mRFP expression over time by the AR reporter consisting of plldR+Switch +mRFP at different lactate concentrations.</figcaption>
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Latest revision as of 07:19, 12 October 2022


pLldR-mRFP

pLldR-mRFP consists of the pLldR promoter ( BBa_K4156101 ) and a downstream RFP sequence. pLldR-mRFP was designed to test the performance of the pLldR promoter by detection of a red fluorescent signal.


Usage and Biology

In this BioBrick, we used the pLldR complex promoter to regulate the expression of the downstream mRFP and eventually introduced the rrnB T1 terminator. this design allowed the spatial and temporal intensity of gene expression under the pLldR promoter to be measured by the red fluorescent signal. We were thus able to verify the specific response of the pLldR promoter to lactate.

Characterization

lactate induced promoter testing

We constructed a lactate reporter consisting of the lactate-inducible promoter pLldR+mRFP. To test its performance, we added reporter in different chassis organisms.Fig 1 indicates that pLldR induces the expression of the downstream gene mRFP with the decrease of O2. Thus, it can be seen that the lactate reporter can work properly.

control
Figure2:Induction of downstream gene mRFP expression over time by the AR reporter consisting of plldR+mRFP 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 680
    Illegal AgeI site found at 1908
    Illegal AgeI site found at 2020
  • 1000
    COMPATIBLE WITH RFC[1000]