Difference between revisions of "Part:BBa K4156114"
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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 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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To improve signaling stability as well as accuracy, we added Amplifying genetic switches based on serine integrase (Bxb1,TP901) to the R reporter(BBa_ ) to construct the AR reporter. 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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                <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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                 <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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                 <figcaption><b>Figure2:</b>Induction of downstream gene mRFP expression over time by the AR reporter consisting of plldR+mRFP at different lactate concentrations.</figcaption>
 
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>

Revision as of 14:29, 10 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

In vitro characterization and data analysis of the reported strains

To improve signaling stability as well as accuracy, we added Amplifying genetic switches based on serine integrase (Bxb1,TP901) to the R reporter(BBa_ ) to construct the AR reporter. 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.

control
Figure 1: Induction of downstream gene mRFP expression over time by the AR reporter consisting of plldR+Switch +mRFP at different lactate concentrations.

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]