Part:BBa_K2991014:Experience

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Applications of BBa_K2991014

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I - Testing of the functionality of our designed parts

After transforming K12 MG1655 E.coli with our pARA-CFP construct, we measured by spectrofluorometry the fluorescence of the transformed bacteria cultivated in M9 medium in the presence of saturating concentration (0.2%) of the associated sugar. The fluorescence studied in the cases below has been normalized with the Optical Density (OD).

Our spectrofluorometric measurements were carried out on the transformed bacteria cultivated in a M9 medium in 2 different conditions : with 0.2% of the specific sugar, and without sugar.

● pARA-CFP: E.coli :

Figure 2:CFP Fluorescence normalized with OD for E.coli K12 MG1655 transformed with pARA in the presence (cyan) or absence (grey) of arabinose at 0,2%. Experiments were conducted during 17 hours.

The activity of pARA is relative to the expression of CFP fluorescence. In the condition “without sugar”, there is no increase in CFP. There is therefore no promoter activity. This validates our control. In the presence of 0.2% arabinose, we observe a strong increase in CFP expression starting in the second hour of the bacterial culture. The CFP in this condition is significantly different than the CFP in the absence of sugar. This verifies the proper functioning of our designed pARA promoter.

Summary

pARA is significantly activated in the presence of arabinose compared to the condition without sugar, verifying the proper functioning of this part.

II - The link between sugar concentration and promoter activity

For these tests we measured the fluorescence in K12 MG1655 E.coli transformed with each of our pARA-CFP construct. The bacteria were cultivated in M9 medium with various different concentrations of the associated sugar. The fluorescence studied in the graphs below has been normalised with the Optical Density (OD).

● pARA-CFP E.coli :

Figure 10:CFP Fluorescence normalized with OD for E.coli K12 MG1655 transformed with pARA in selected concentrations of arabinose

The activity of pARA is relative to the expression of CFP fluorescence.
Without sugar, we observe a very low level of CFP fluorescence which allows us to validate the proper functioning of our pARA promoter in this condition. The difference of CFP expression between conditions with arabinose and the condition without sugar becomes significant around the fourth hour of bacterial culture.
From 14.5h, we notice a significant difference of CFP expression amongst the conditions containing 0.1 %, 0.05% and 0.01% of arabinose. However, the curve of the condition containing 0.02% of arabinose follows the same tendency as the previous curves, but it has enormous error bars. If we consider the results of this condition to be distorted, we can conclude that the intensity of the activation of pARA can be regulated proportionally to the concentration of arabinose in the medium

Summary

In our simple insert construct, we showed that pARA is the only promoter that responded to the concentration level of its cognate sugar.

III - Binary combinations of sugars with simple and double inserts

Here we tested the activities of single insert pARA-CFP with binary combinations of sugars. The primary purpose of this experiment is to analyse the activity of the pARA promoter in the presence of 2 sugars at a time: it’s own specific sugar and another. We will see if the sugar-hierarchy is still present in our construction in this condition, and how a sugar, higher or lower in the hierarchy impacts the expression of the promoter.

● pARA-CFP E.coli :

As a reminder, here E.coli K12 MG1655 was transformed with pARA-CFP. We monitored pARA activity by measuring CFP fluorescence in absence or presence of combinations of sugars. Results are provided below (Fig. 23)

Figure 23: CFP Fluorescence normalized with OD for E.coli K12 MG1655 transformed with pARA-CFP in absence of any sugar (■), in the presence of 0.2% arabinose combined with M9 rich medium (●), 0.2% lactose (○), 0.2% sorbitol (△) and 0.2% ribose (+). Experiments were conducted during 17 hours.

No change in CFP fluorescence is observed in the absence of sugar. This validates our negative control.
Contrasting results were obtained for the different combinations of sugars. In presence of saturating arabinose (0.2%) and M2 rich medium, CFP fluorescence slightly increased over time with a fluorescence level that was slightly above that of the negative control after 17 hours. A similar pattern is observed when 0.2% arabinose is combined with 0.2% lactose. This would suggest that M9 rich medium likewise lactose would have an inhibitory effect on pARA activity. Indeed, when arabinose is combined with others sugars lower in the hierarchy, i.e sorbitol or ribose, the activity of pARA promoter was much higher. With sorbitol, the CFP fluorescence increased steadily between 5 hours and 10 hours of culture to reach a plateau after the 10th hour. When combined with 0.2% ribose, the fluorescence pattern was different as it exhibited a steady linear increase over time.
These results show that ribose and sorbitol have a cumulative effect with arabinose that leads to a bigger activation of the pARA promoter.

Summary

We can conclude that in this simple construct, a sugar higher up in the hierarchy will inhibit promoters associated to sugars lower in the hierarchy. For example, when pARA is placed in the presence of 0,2% arabinose and 0.2% lactose, pARA is not activated. Lactose, being higher up in the hierarchy than arabinose, inhibits pARA.
A sugar which is lower in the hierarchy has no effect on the activity of a promoter associated with a sugar of higher hierarchy.

IV - Duration of the promoter expression

As our tool is mainly based on controlling gene expression in time, it is important for us to study the activity of this promoter in time. This will allow us to better understand the behavior of pARA promoter, enabling us to take into account any time differences we might discover in the design of our tool.

● pARA-CFP E.coli :

Figure 28:Rate of CFP Fluorescence/hours for E.coli K12 MG1655 transformed with pARA-CFP simple insert in the absence or in the presence of 0,2% of Arabinose

The activity of pARA is relative to the expression of CFP fluorescence.
We do not observe an increase in the rate of production of CFP fluorescence in the condition without sugar. It confirms our control. With 0,2% of Arabinose, we observe a steady increase of the CFP production rate beginning at hour 1,5. At hour 4,5 the rate of CFP production reaches a plateau and remains in this state throughout the end of the experiment.
We can conclude that the activity of the promoter pARA starts around 4,5 hours after the presence of sugar, and the activation remains constant.

Summary

We can conclude that the activation and the activity of pARA promoter vary in time, from one to the other. In saturated sugar pARA is activated around hour 4,5 after the addition of sugar, and the activity withheld throughout the end of the experiment.
This information would need to be taken into account to increase the precision of sequential gene expression in time of our tool.