Part:BBa_K3463023:Experience

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Characterization of BBa_K3463023

To confirm the influence of the optical density on the lsrA promoter, we compared the fluorescent expression of mCherry due to a constitutive J23100 and PlsrA.

For this experiment all the construction PUCBB-J23100-mCherry, pUCBB-PlsrHigh-mCherry (BBa_K3463023 )used were successfully obtained and transformed in E. coli BL21

Each bacteria was cultivated at 0,5 OD in LB, we measured the optical density and the mCherry fluorescence hourly (figure 1) . An Opaque-walled 96 was used with the FluoStar BMG labtech and the fluorescent gain had been set to 2000 (we made triplicate measurement). This experience was repeated three times in the same conditions.

Figure 1:A

Figure 1:B mCherry fluorescence expression reported at bacterial optical density over time.To represent the two curves, we used two gradations: the one on the left represents the expression of the lsr promoter and the one on the right represents the J23100 promoter. A and B are two examples of the data obtained in the same working conditions

Figure number 1 shows the expression of the mCherry under PlsrA, J23100 promoter and BL21 WT as control over time.

We can first observe that the expression of mCherry under the control of PlsrA occurs between 7 and 8 hours after inoculation, while the constitutive promoter expresses mCherry earlier (after only 3 to 4 hours). In addition, the level of expression of mCherry is about 10 times lower under the control of PlsrA rather than under the control of the constitutive promoter J23100 which informs about the strength of the promoter.

Intending to prove that the Plsr expression is related to Autoinducer 2 (AI-2) we performed the same experiment with E. coli DH5 alpha. This strain is known for not producing any AI-2, and as expected there was no mCherry expression during the growing time (results not shown).

We also calculated the Spearman correlation between fluorescence and optical density. For the constitutive promoter we obtained 0.6263641 and for PlsrA 0.4615318. A higher correlation is observed for J23100 which is explained by a constant expression during growth unlike the expression of the lsr promoter.

The delay of expression observed with lsr promoter in BL21 relative to the constitutive promoter J23100 is probably linked to the necessary accumulation of Ai-2 during growth and therefore to the optical density. Although this experiment was repeated several times under the same conditions we did not obtain the same results as we can notice (figure 1). Even if the trend suggests that the expression of the lsr promoter is linked to the optical density, there are probably other factors we have involuntarily stimulated that could influence the expression of the protein.

Comparison between BBa_K3463023 and BBa_K34630024 expression

mCherry fluorescence expression under control of plsr reported at bacterial optical density over time.

We see that the expression of plsr_high and plsr_low is simultaneously triggered around 7.5h. It can also be noted that the difference between the two becomes more and more pronounced. Each time we reproduced this experiment with E.coli BL21 we always found a significant difference in expression according to the UTR sequence.

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