Difference between revisions of "Part:BBa K3183100"

Revision as of 14:39, 21 October 2019

Erythromycin Promoter Reporter Gene

Erythromycin Promoter fused with mGFP5

Sequence and Features

Characterisation

Reporter of constitutive expression in L. reuteri

Summary
We have used this part as a reporter of transformation success in our work on L. reuteri, and as a positive control for protein expression.
Methods
The composite part was inserted into the pTRKH3 vector by Gibson Assembly and transformed into L. reuteri 10023c by electroporation. The transformants were used in a fluorometric assay using excitation at 500 nm and detecting emission at 520 nm; the assay was used to show the relationship between exogenous protein expression and bacterial growth rate by comparing the OD600 and relative fluorescence of wild type and transformed bacteria. In addition, the part was used in fluorescence microscopy using the same absorption and emission wavelengths to determine the cytoplasmic protein distribution/morphology:
Results:

Fig. 1: Transformant Fluorescence: Normalized fluorescence of the cell cultures was determined by calculating the ratio of raw fluorescence to the optical density at 600 nm to ensure that the fluorescence levels measured are a result of reporter gene expression and not simply due to cell growth. The observed drop in fluorescence/OD600 across the time period can be accounted for by considering the large background fluorescence signal of MRS, such that the fluorescence stays nearly constant, while the OD increases markedly as a result of cell proliferation.

Fig. 2: Fluorescence Microscopy: top row: micrographs of normalised exposure show the relative levels of exogenous protein expression in 3 strains of Lactobacillus reuteri 100-23c: wild type, pTRKH3-erm-GFP and pTRKH3-erm-slpMod CD27L_mClover. Bottom row: the corresponding bright field imaging mode. As expected, no fluorescent protein expression is detected in the wild type strain, while significant levels are observable in the GFP transformants. However, the CD27L shows low level expression concentrated in inclusion body-like structures.

Measurement of promoter strength

Summary
Another 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 3: 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 outliar due to random error in the instrument. 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 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 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 the concentration of nutrients in the medium decreases which limits protein expression inside the cell. Error bars represent 1 standard deviation. n = 3

Discussion:
The results section shows that the blanc corrected fluorescence intensity signals often have negative values. 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 most frequently led to negative values. -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 fluors, 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.