Part:BBa_K3183000
Erythromycin Constitutive Promoter
P-erm is a constitutive promoter which can be used in Lactobacillus reuteri 10023C, and may have uses in other Lactobacillus species. It has also been shown to be functional in E. coli.
The promoter is derived from the erythromycin ribosomal methylase (ermB) promoter from the broad-host range plasmid pAMβ1 isolated from Enterococcus faecalis.1 It was subsequently characterised in six strains of Lactobacillus reuteri and Lactococcus lactisspp. cremoris MG1363.2
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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:
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:
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.
Characterisation
This part was characterised in the composite part BBa_K3183100. Data for this part is shown below.
References
1. Swinfield, Tracy-Jane, et al. “Physical Characterisation of the Replication Region of the Streptococcus Faecalis Plasmid pAMβ1.” Gene, vol. 87, no. 1, 1990, pp. 79–90., doi:10.1016/s0378-1119(19)30488-3. 2. Lizier, Michela, et al. “Comparison of Expression Vectors in Lactobacillus Reuteri Strains.” FEMS Microbiology Letters, vol. 308, no. 1, 2010, pp. 8–15., doi:10.1111/j.1574-6968.2010.01978.x.
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