Team:Lambert GA/Results
Results
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 tsPurple Data
As predicted, hue values decreased as IPTG concentrations increased with only slight variation. This variation is found in the 500 uM IPTG samples, which had a value of 100% purple, and is greater than the recorded 99.5% purple value in the 1000 uM IPTG tube. This variation may be an error originating from when pictures were taken using the Chrome-Q. Despite this miscalculation, the 500 uM IPTG was recorded 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. (Note: the values where hue equals 400.2 correlates with 40.2 degrees, since values over 360 degrees indicates lighter coloration, such as the “yellow” found in non-expressive E.coli). As for results, the calculated mean was 316.1 uM IPTG, which indicates at what IPTG level the highest percentage of purple will occur. The other values measured were luminance values, which indicated the amount of light emitted or reflected. These results reveal that as IPTG concentrations increase, luminance values decrease. This portion of the data confirmed the expected results since the lighter cell color found in non-expressive “yellow” cells emits a greater light intensity than the purple found in IPTG induced cells. An important point to note is that the values shown in the table and graphs are averages from rows of triplicates to reduce the effects of variability. Overall, the results from tsPurple and the Chrome-Q app indicated a negative correlation between hue and IPTG concentration, however more trials should be completed to further standardize the tsPurple results.
Scrooge Orange and Virginia Violet
The data collected from the Scrooge Orange and VirginiaViolet trials were congruent with the hypothesis. Hue values for Scrooge Orange, although lesser in intensity than the data calculated for the tsPurple and VirginiaViolet trials, decreased as IPTG values increased. Similarly, luminance values decreased as IPTG concentrations increased along with a reduction of variability in Virginia Violet and ScroogeOrange trials when comparing this data to the tsPurple trials. Standard deviation values demonstrated a relatively low variability throghout the data. There also presented a negative correlation between IPTG concentrations and hue values.
Conclusion
In order to appropriately characterize our proteolysis, we conducted an additional study to serve as a proof of concept. The ATUM Protein Paintbox allowed us to verify the changes in hue and percent color upon induction with IPTG. The induction of the cells with IPTG induced additional expression of the chromogenic protein due to the inhibition of LacI’s repressor proteins, allowing for T5 to continuously express the chromoprotein. There was direct positive correlation between IPTG concentration and the percent color expressed by the cells, and a direct negative correlation between IPTG concentration and hue. This trend visualized between hue and IPTG can be visualized using the RGB color space. The specific RGB values are quantified in degrees between 0 and 360, and the Hue is calculated using an algorithm we developed. Although the RGB values are visualized in a cylindrical space, the specific Hue value is obtained from a specific point value, allowing the trend to be validated. As IPTG concentration increased, the T5 expression allowed for an increase in the chromogenic protein concentration, leading to an increased expression of color, verifying the increase in percent color. In addition, our usage of both tsPurple and Virginia Violet proved that despite the similarity in color, there is a substantial difference in hue and percent color across induction levels between the two chromoproteins. Through this, we were able to prove the concept of chromoprotein expression across various levels of IPTG Induction.
For the construct we created, consisting of the tsPurple with a constitutive promoter and tsPurple-LAA with a constitutive promoter, we were additionally able to visualize the difference in chromoprotein expression. As the LAA is an SsrA degradation tag, the tsPurple LAA was likely targeted by naturally occurring ClpXP proteins, allowing for the degradation of the purple chromoprotein. This led to almost all purple color being absent in the liquid culture, verifying the occurrence of the protein degradation. On the other hand, the tsPurple with no degradation tag was constitutively expressed, allowing for complete color expression within the cultures. These were analyzed in comparison to regular E. Coli cells, and the hue/percent color values for the tsPurple-LAA were almost analogous to the regular E. Coli cells, while the tsPurple with no degradation tag had high hue and percent color values, indicating the functioning of the ClpXP mechanism occurred successfully.
Through the comparison of our experimental data with our proof of concept, we were able to successfully discern a variation in hue/percent color values with different levels of protein expression and degradation, serving as an effective measurement model. Through the usage of our Chrom-Q prototype, we were able to quantify these variations in values using an alternative model at a low-cost, thereby having the potential to serve as a new basis of measurement in underfunded labs without access to plate readers.
Furthermore, the presence of a negative correlation between IPTG concentrations and luminance/ hue values indicates a quantified trend in chromoprotein expression. The accuracy of the engineered Chrome-Q system is also verified in the differences found in the magnitude of calculated hue and luminance values for each chromoprotein, where tsPurple and VirginiaViolet had the highest mean hues/luminance values, while Scrooge Orange had the lowest mean hue/luminance values. Overall, the Chrome-Q system is a valid system when quantifying and distinguishing chromoprotein color expression.
