Part:BBa_K3183010

mClover3 Fluorescent Protein, Codon Optimized for L. reuteri

mClover3 is a green fluorescent protein derivative which has been codon optimized for Lactobacillus reuteri 10023C, and may have uses in other Lactobacillus species. mClover3 is a 26.9 kDa protein derived from GFP. mClover3 improves photostability by 60% (t_1/2 = 80s) to its predecessor, owing to 2 mutations relative to dClover2: A206K and S160C. mClover3 can be used in Förster Resonance Energy Transfer (FRET) experiments with mRuby3, providing an alternative to cyan/yellow partners. This has the advantage of reducing spectral separation, having lower phototoxicity, and lower autofluorescence¹.

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]

Fluorescence wavelengths

Bajar et al report the following excitation and emission data for mClover3 -

• Excitation max - 506nm
• Emission max - 518nm

Figure 1: mClover3 spectrum

Parts characterized by Oxford iGEM 2019

This part was characterised in the composite part BBa_K3183028, and BBa_K3183101.

The purified protein (BBa_K3183203) was used to make a standard curve for mClover3 with a fluorometer. We used two different buffers: phosphate buffered saline (PBS) and De Man, Rogosa and Sharpe (MRS) media.

Figure 1: mClover3 standard curve - Fluorescence intensity vs Concentration (mg/ml). On the graph, we can observe that in MRS the FI is lower than that in PBS. This could be due to the high fluorescence of MRS that masks mClover3 fluorescence.Error bars represent 1 s.d., n=3

The protein mClover3 was mainly used as a reporter. It enables measurement of promoter strength.

Measurement of promoter strength: BBa_K3183028 and BBa_K3183101

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: 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: erm promoter FI and OD600 time dependence - Blank corrected Fluorescence intensity and OD600 was plotted against time for erm promoter. From this graph, we can observe that the rate of expression of mClover3 decreases over time as does the growth. Error bars represent Standard error of the mean. n = 3

Figure 3: 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.

Use by Team Oxford 2019

This part was used in the following composite parts: BBa_K3183028, BBa_K3183300, BBa_K3183104, BBa_K3183203, BBa_K3183101, and BBa_K3183104.

References

1. Bajar, Bryce T., et al. “Improving Brightness and Photostability of Green and Red Fluorescent Proteins for Live Cell Imaging and FRET Reporting.” Scientific Reports, vol. 6, no. 1, Feb. 2016, p. 20889. DOI.org (Crossref), doi:10.1038/srep20889.