Part:BBa_K3183028

erm-mClover3 codon optimized for L. reuteri

This is reporter gene compromised of P-erm and mClover3 fluorescent protein. The reporter gene is designed for L. reuteri but it works in E. coli as well. The reporter gene produces mClover3 (BBa_K3183010) if P-erm (BBa_K3183000) is active in the cell. This activation can be measured by fluometric assay.

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
  COMPATIBLE WITH RFC[1000]

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 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: erm promoter FI and OD600 time dependence - Blank corrected Fluorescence intensity and OD600 was plotted against time for ldh promoter. From this graph, we can observe that the rate of expression of mClover decreases over time as does the growth. 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.