Part:BBa_K3827002
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, a synthetic promoter, and lldR, RFP, and two terminators. LldR is constitutively expressed, through the synthetic promoter. This allows lldR to continuously bind to the pLactate, negatively regulating the promoter, until lactate is present. Lactate then binds to the lldR, preventing it from repressing pLactate, and allows for transcription of the genes downstream pLactate to be transcribed.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1018
Illegal NheI site found at 1041 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 773
Illegal AgeI site found at 885 - 1000COMPATIBLE WITH RFC[1000]
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Characterization
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.
Results
Fig. 4 A Data from our lactate induction experiment with our lactate inducible reporter construct, with the OD600 (cell density) plotted against time. We discarded the 0 h time point data due to technical artifacts caused by bubbles in the plate reader wells leading to a spike in the OD600 measurements. Looking at the 2h time point (second read), the bubbles have disappeared, removing the obstruction towards measuring the OD600 of the cells. Cell growth in media with a lactate concentration of 100 nM is noticeably lower than the rest of concentrations. This matches the literatures’ reports of lactate poisoning at higher concentrations in E. coli.
Fig. 4 B and C Data from our lactate induction experiment with our lactate inducible reporter construct, with the lactate concentrations plotted against Normalized fluorescence. As expected, 0 h has the lowest fluorescence, especially since the cells haven’t had time to acclimate to the presence of lactate in the media. Overall, there is a pattern of increased fluorescence with an increase of lactate concentration, with a particularly steep increase in the concentrations between 0 and 25 mM seen in plot C. Unsurprisingly, the steep increase at the lower concentrations match the kinetics of a similar lactate operon found in E. coli which mirrors a hill function. The fluctuations between hours may be due to technical artifacts and the large 2 hour time span between each read, especially at hour 2. However, as seen in plot B, there is still an overarching increase of fluorescence over time with most of the times later in the experiments expressing higher amounts of fluorescence.
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