Difference between revisions of "Part:BBa K3827002"
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<h1>Characterization</h1>
 
<h1>Characterization</h1>
 
[[File:plac_lldr_rfp_construct.jpg|700px|thumb|center|Lactate induction experiment of pLactate promoter in combination with lldR and a RFP gene at different lactate concentrations over a period of 18 hours.]]
 
[[File:plac_lldr_rfp_construct.jpg|700px|thumb|center|Lactate induction experiment of pLactate promoter in combination with lldR and a RFP gene at different lactate concentrations over a period of 18 hours.]]
 +
 +
<p> As the lactate inducible promoter gets induced once lactate binds to its substrate, we measured the promoter's activity by adding different concentrations of lactate in a plate reader. </p>
 +
 +
<p>Day before the experiment was run:</p>
 +
<p>Cells with constitutively expressed RFP cells (positive control), cells with an empty plasmid (negative control), and cells with our construct were inoculated at 37C overnight.
 +
Lactate solutions were diluted to the appropriate concentrations to be put in the reader (0mM, 2mM, 5mM, 15mM, 40mM and 100mM lactate). These concentrations were determined through literature searches of lactate presence in healthy and tumorous human tissue.</p>
 +
<p> Day of the experiment: </p>
 +
<p> The OD600 of the cells was diluted to 0.5 OD600 (and checked using the spectrometer) so that the concentration of the cells is correct after being added into the media by the liquid dispenser. </p>
 +
<p> The media with cells was loaded into a 96 plate using a multichannel pipettor, and fluorescence readout measured using plate reader.</p>
 +
 +
<p>From this assay we hoped to validate the promoter’s nature, as it is induced by different lactate concentrations. If the promoter would be working as expected, we would be able to see a bigger RFP expression as lactate concentration increases, and consequently prove that the promoter worked. After running the experiment and collecting the data, we were able to see the promoter getting induced by seeing increasing levels of RFP being expressed as the concentration of lactate increased, successfully achieving proof-of-concept experimental results. </p>

Revision as of 05:38, 20 October 2021


Lactate Inducible Reporter (pLactate + lldR + RFP)

This is a composite reporter construct made to characterize the activity of the pLactate (lactate inducible promoter) and lldR (regulatory protein) unit. It consists of the pLactate, RBS, lldR, RFP, and a terminator.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1018
    Illegal NheI site found at 1041
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 773
    Illegal AgeI site found at 885
  • 1000
    COMPATIBLE WITH RFC[1000]


Characterization

Lactate induction experiment of pLactate promoter in combination with lldR and a RFP gene at different lactate concentrations over a period of 18 hours.

As the lactate inducible promoter gets induced once lactate binds to its substrate, we measured the promoter's activity by adding different concentrations of lactate in a plate reader.

Day before the experiment was run:

Cells with constitutively expressed RFP cells (positive control), cells with an empty plasmid (negative control), and cells with our construct were inoculated at 37C overnight. Lactate solutions were diluted to the appropriate concentrations to be put in the reader (0mM, 2mM, 5mM, 15mM, 40mM and 100mM lactate). These concentrations were determined through literature searches of lactate presence in healthy and tumorous human tissue.

Day of the experiment:

The OD600 of the cells was diluted to 0.5 OD600 (and checked using the spectrometer) so that the concentration of the cells is correct after being added into the media by the liquid dispenser.

The media with cells was loaded into a 96 plate using a multichannel pipettor, and fluorescence readout measured using plate reader.

From this assay we hoped to validate the promoter’s nature, as it is induced by different lactate concentrations. If the promoter would be working as expected, we would be able to see a bigger RFP expression as lactate concentration increases, and consequently prove that the promoter worked. After running the experiment and collecting the data, we were able to see the promoter getting induced by seeing increasing levels of RFP being expressed as the concentration of lactate increased, successfully achieving proof-of-concept experimental results.