Part:BBa_K3183101

Lactate Dehydrogenase Promoter Reporter Gene

Summary

This part comprises the basic parts BBa_K3183002 and BBa_K3183010; its purpose was to enable the characterization of lactate dehydrogenase promoter (BBa_K3183002) in comparison to other L. reuteri promoters (cf. BBa_K3183028). This is achieved based on the assumption that the cell's detected fluorescence is proportional to the fluorescent reporter's concentration, which, in turn, reflects the strength of the promoter.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 219
  

Part characterization by Oxford iGEM 2019

Measurement of promoter strength

Summary
A major use of this part was to facilitate the quantification and comparison of promoter strengths in vivo. The principle of such an assay is to correlate the fluorescence intensity of our bacterial sample to the fluorescence intensity of a fluorescein solution of known concentration, thus allowing us to estimate the exact protein concentration under the control of the promoter reached in the cytoplasm.

Method:

The composite part was inserted into pTRKH3 vector BBa_K3183050 by Gibson assembly and transformed into E.coli by heat-shock transformation. Successfully transformed colonies were picked and used in fluorometric assay using excitation at 500nm and detecting emission 520nm. The assay was used to compare the protein expression strength of the two promoters by measuring fluorescence intensity and OD600 over time. Then, to normalize the results, the blank corrected ratio of fluorescence intensity and absorbance at 600nm was used to compare the promoters.
Results:

Figure 1: ldh promoter FI and OD600 time dependence - Blank corrected Fluorescence intensity and OD600 was plotted against time for ldh promoter. A large peak in OD600 can be observed, which could be an outlier due to measurement error. Error bars represent Standard error of the mean. n = 3

Figure 2: Promoter strength comparison - The blank corrected fluorescence intensity and OD600 ratio was plotted against time for both promoters. On the plot, the mean of ldh promoter seems to be larger than that of erm promoter. However, due to the broad standard deviation, no significant conclusion can be made. On the other hand, for erm promoter, it could be observed that the FI/OD600 decreases over time. One hypothesis is that due to the cell growth (increased OD600) and increased scattering, the fluorescence intensity decreases. Error bars represent 1 standard deviation. n = 3

Discussion:
The results section shows that the blank corrected fluorescence intensity have very high standard deviations. This is likely because, instead of purifying the protein and exchanging the buffer, we performed our assays on living cells; this had a number of consequences on the accuracy of our results:

The MRS medium in which the cells were grown has very high background fluorescence, such that its intrinsic noise significantly overshadowed the signal and sometimes lead to unreasonable results.

The optical density of the solution due to light scattering by bacteria led to a significant drop in signal intensity, which would have been extremely difficult to correct for at large ODs

The vastly different chemical properties (e.g. ionic strength, the presence of quenchers etc.)of the cytosolic environment from regular buffer solutions likely result in very different spectroscopic properties of the fluorophores, such as quantum yield and maximal absorption/emission wavelengths, thus reducing the feasibility of comparison of our sample to the calibration curve based on fluorescein.

Therefore, we argue that the data we obtained cannot be used to quantitatively assess the strength of the promoters and has, at most, qualitative value. Therefore, we suggest that in the future more rigorous assays performed by purifying the enzyme and measuring its fluorescence after the buffer was exchanged to one similar to that of the fluorescein solution.