Difference between revisions of "Part:BBa K3183101"

Revision as of 16:15, 21 October 2019

Lactate Dehydrogenase Promoter Reporter Gene

Summary

This part comprises the simple parts BBa_K3183002 and BBa_K3183010; its purpose was to enable the characterization of lactate dehydrogenase promoter (BBa_K3183002) in comparison to other L. reuteri promoters (cf. BBa_K3183028). This is achieved based on the assumption that the cell's detected fluorescence is proportional to the fluorescent reporter's concentration, which, in turn, reflects the strength of the promoter.

Sequence and Features

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