Difference between revisions of "Part:BBa K3183028"
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<partinfo>BBa_K3183028 short</partinfo> | <partinfo>BBa_K3183028 short</partinfo> | ||
| − | This is reporter gene compromised of P-erm and mClover3 fluorescent protein. | + | This is reporter gene compromised of P-erm and mClover3 fluorescent protein. The reporter gene is designed for <i>L. reuteri</i> but it works in <i>E. coli</i> as well. The reporter gene produces mClover3(<partinfo>BBa_K3183010</partinfo>) if P-erm (<partinfo>BBa_K3183000</partinfo>) is active in the cell. This activation can be measured by fluometric assay. |
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<partinfo>BBa_K3183028 parameters</partinfo> | <partinfo>BBa_K3183028 parameters</partinfo> | ||
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| + | ===Part characterization by Oxford iGEM 2019=== | ||
| + | <b> Measurement of promoter strength</b><br> | ||
| + | <br><b> | ||
| + | Summary | ||
| + | </b><br> | ||
| + | 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. | ||
| + | <br><b> | ||
| + | Method: | ||
| + | </b><br><br> | ||
| + | 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. | ||
| + | <br><b> | ||
| + | Results: | ||
| + | <br><br></b> | ||
| + | [[File:T--Oxford--Results-ldh.png|thumb|left|430px| | ||
| + | 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. <i>Error bars represent Standard error of the mean. n = 3</i> ]] | ||
| + | [[File:T--Oxford--Results-erm.png|thumb|right|430px| | ||
| + | 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. <i>Error bars represent Standard error of the mean. n = 3</i> ]] | ||
| + | <br><br> | ||
| + | |||
| + | [[File:T--Oxford--Results-erm-vs-ldh.png|thumb|center|430px| | ||
| + | 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. <i>Error bars represent 1 standard deviation. n = 3</i>]] | ||
| + | |||
| + | <b>Discussion</b> | ||
Revision as of 15:50, 21 October 2019
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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE 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:
Discussion