Team:Lambert GA/Results

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Conclusion and Results

Expected results correlate with the genetic design found on the Experiments page. To recapitulate, the full construct contained the LacI repressor system, which initially impeded the promotion of sequence, ClpXP CI, located downstream of pLac; upon IPTG induction, LacI, when encountered by the chemical, exhibited repression as IPTG molecules bound to Lac repressor proteins. This inhibition allowed promotion of the latter portion of our construct: pLac ClpXP CI. Because of ClpXP’s activation, the transcribed protein was able to recognize the middle portion of our construct containing an SsrA tag attached to tsPurple and, thereby, degrade our reporter chromoprotein. In addition, the CI (competitive inhibitor), was able to bind and inhibit pλR, obstructing the promotion of tsPurple. Therefore, the working construct would supply us with a concentration of translated tsPurple dependant upon the strength of the attached degradation tag (LAA, DAS or no tag present); color expression would then persist inconstancy as the CI repressed the excess promotion of tsPurple. Following the workflow, we then would have transformed our constructs into varied Keio Knockout and Wild strains in order to validate the functional portions of ClpXP. Concluding the institution of our genetic parts into Keio strains, the inoculation of colonies - using the refined methodology demonstrated in our Model and Demonstrate pages- into triplicate trials of 0M, 10 uM, 100 uM, 500 uM, and 1mM concentrations of IPTG, with antibiotic Luria broth as our solvent, would provide us with enriched data to then utilize our Chrome-Q. From calculated HSV color values, it is predicted that as IPTG concentration increase then the hue values will decrease. Hue is measured from 0 to 360 degrees; lower magnitude hue values indicate darker spectrum colors and higher magnitude hue values indicate lighter spectrum colors. Although we are using the HSV color model we decided to omit saturation values. Because saturation measures opacity/ transparency and the data collected will range from a purple to yellow (non-expressive cells) coloration, then calculations of opacity are ineffectual. Finally, value, synonymous to the term “luminance” is also predicted to decrease as IPTG concentrations increase. Luminance indicates emitted light intensity in terms of the amount of light absorbed (low luminance values) and emitted (high luminance values). Because the yellow coloration, observed in non-expressive cells are "brighter" and emit more light than the purple, IPTG induced cells, than it is predicted luminance values will decrease as IPTG levels increase.

As lab work concluded the proposed construct could not be successfully assembled. The initial portion of the construct: pλR LacI, yielded low miniprep concentrations that were impractical to proceed within the cloning workflow. We then attempted to ligate pλR B0034 LacI, but gel results, determining the existence of the sequences, were inconclusive. Because numerous experiences with the promoter, pλR, had not delivered applicable results, the R0040 promoter, obtained from the Interlab Study, was instituted as a replacement. Despite the initial obstacle, the middle parts of the proposed construct, being tsPurple and increasingly potent degradation tags, were ordered from IDT and successfully underwent the cloning procedure. The parts: tsPurple, tsPurple DAS, and tsPuple LAA, were sent for sequencing. The sequence containing LAA, and the sequence without a degradation tag both were verified as complete and void of mutations; the sequence containing DAS was received as lacking the degradation tag. The latter portion of the construct, composed of pLac ClpXP CI, was successfully assembled and sent for sequencing. Results from sequencing concluded that CI had mutated and, therefore, not functional. From the corroborated cloning parts, the sequences containing LAA and no degradation tags were individually ligated to the R0040 promoter and RBS. The ligations were then transformed into competent, NEB 5-Alpha cells, plated and visualized the next day. The presence of purple coloration verified the success of ligation. Despite lacking the full, genetic construct, we managed to obtain viable data from the Chrome-Q system using the proof of concept.

Proof of Concept utilized ATUM’s Protein Paintbox with the chromoproteins: TinselPurple (tsPurple), ScroogeOrange, and VirginiaViolet, and successfully underwent the optimized protocol. The resulting color expression, correlating with incremental concentrations of IPTG, was analyzed using the Chrome-Q system. Methodology for the Chrome-Q system can be found on the Model page. The data obtained was only calculated for tsPpurple induced cells:

Concentration is measured in microliters (uL). Hue is measure from 0 to 360 degrees , and saturation and value are measured from a scale, 0-100. Saturation values were not calculated because the mobile app’s purpose was to accurately calculate color percentage. Because saturation measures opacity/transparency, and the colors visualized from the cells ranged from a yellow hue (0M IPTG) to dark purple (1mM IPTG) then measurements of opacity/ transparency are ineffectual. Luminance is calculated using the HSV color space.

Analysis of Data

As predicted, hue values decreased as IPTG concentrations increased with only slight variation. This variation is found in the 500 uM IPTG tube where the calculated, 100% purple, is greater than the 99.5% purple in the 1000 uM IPTG tube. This variation may be an error when we were taking the picture against the Chrome-Q. The calculated mean was 316.1 uM IPTG, which indicates when the highest percentage of purple will occur. Despite this calculation, we labeled the 500 uM IPTG as 100.0% purple because this data point represents the relative percentage of purple against the total set of data. Data representing 100% purple indicates the minimum hue value and data representing 0.0% purple indicates the maximum hue value. Where hue equals 400.2 correlates with 40.2 degrees (values over 360 degrees indicates lighter coloration, such as the “yellow” found in non-expressive E.coli). Luminance values, indicating amount of light emitted or reflected, reveal that as IPTG concentrations increase, luminance values decrease. This portion of the data was correctly hypothesized as the lighter cell color found in non-expressive “yellow” cells emits a greater light intensity than the purple found in IPTG induced cells. Because the data calculated was not repeated in triplicate, we cannot confirm that the the mobile app accurately represents color percentage. Rather, it can be deduced that the mobile app represents the color percentage as there is a perceived negative correlation between hue and IPTG concentration. In order to validate this assumption, trials containing the tsPurple constructs must be repeated. Further, standardization of results must be calculated to perfect the Chrome-Q.