Difference between revisions of "Part:BBa K3183000"
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A major use of this part was to facilitate the quantification and comparison of promoter strengths <i>in vivo</i>. 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.
 
A major use of this part was to facilitate the quantification and comparison of promoter strengths <i>in vivo</i>. 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.
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Method:
 
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The composite part was inserted into pTRKH3 vector by Gibson assembly and transformed into <i>E.coli</i> 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.
 
The composite part was inserted into pTRKH3 vector by Gibson assembly and transformed into <i>E.coli</i> 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.
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Results:
 
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[[File:T--Oxford--Results-ldh.png|thumb|left|430px|
 
[[File:T--Oxford--Results-ldh.png|thumb|left|430px|
 
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 outliar due to random error in the instrument. <i>Error bars represent Standard error of the mean. n = 3</i> ]]
 
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 outliar due to random error in the instrument. <i>Error bars represent Standard error of the mean. n = 3</i> ]]
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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.  
 
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.  
 
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===References===
 
===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. <br>
 
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. <br>
 
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.
 
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.

Revision as of 20:24, 21 October 2019


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


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]


Characterisation

This part was characterised in BBa_K3183100 and BBa_K3183028. It was also used in the composite part BBa_K3183103. Information on its characterisation is shown below.

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: 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 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 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 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.

    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.