Rhamnose Mediated Characterization

Introduction

The inducible Lac and Tet promoters are repressed by a repressor that can be inhibited by the addition of a chemical inducer. These promoters can be easily characterized by induction with different concentrations of the appropriate chemical. However, this approach is not applicable if the promoter is not inducible by addition of a chemical, such as the CIλ promoter. Therefore, we developed a new system to characterize promoters based on a two plasmid system with a L-rhamnose-inducible promoter (pRha) and green fluorescent protein (GFP) as reporter as represented in Figure 1. Since our Kill-switch system contains the lacI and tetR gene we could not use the IPTG or aTc inducible promoter for regulation of repressor concentration. Therefore, this characterization method could be used for testing and characterizing the toggle switch and total function of the Kill-switch system.

Characterization Design

The final design to characterize promoters includes the following two components:

• A repressor plasmid with a pRha and a repressor gene of choice
• A reporter plasmid with your favoured promoter and GFP

Promoters will be characterized by quantifying the expression of the reporter gene at different concentrations of rhamnose. Therefore, fluorescence will be measured to determine the strength of the promoter. Quantifying fluorescence will indicate how reliable the repressors inhibit the promoter function. We made a collection of BioBricks that can be used for characterization experiments for repressible promoters without the use of repressor protein specific chemicals like IPTG or aTc. The double repressible promoters used in the Kill-switch system are taken from the iGEM Registry. Since these promoters pCI/Lac and pCI/Tet were not characterized we could not estimate if a functional Kill-switch could be constructed with these promoters. The double repressible promoter pCI/Lac from the toggle switch, is characterized. pCI/Lac was combined with a gfp reporter gene under Elowitz RBS (BBa_I13504 ) to make the new BioBrick pCI/Lac gfp . As a reference pRha and the tetracyclin promoter pTet are also assembled to the gfp gene (BBa_K1493501, BBa_K1493504 ). Since the promoter strength of pTet is known in relative promoter unit (RPU) it can be used as a reference promoter to estimate promoter strength [1]. In this way, we can compare the constitutive promoter strength at one time point and give the characterized promoter a RPU value. pRha was successfully assembled to the CI (BBa_P0451) and lac (BBa_P0412) repressor protein genes under Elowitz RBS creating new BioBricks BBa_K1493510 and BBa_K1493520.

Results

The graphs containing all data points can be found in here. The characterization protocol can be found here.

We first characterized pRha itself. The measurements in Figure 2 indicate a tuneable activation of pRha by rhamnose. The RFU values of 0% and 0.001% rhamnose are not significant taking into account the high standard deviation for these measurements as can be seen in Figure 2B. From a rhamnose concentration of 0.01% to 0.2% a significant increase in fluorescence is measured. Fluorescence from figure 2 can be used to predict the concentration of repressor protein produced. Data points for time 8.25 h were chosen for the graph in Figure 2B due to the peak at time for 0.2% rhamnose, which is visible in Figure 2A. This peak can be explained by the rhamnose depletion, as it is consumed by E. coli, causing the stop of promoter induction. We assume that at this time point the highest concentration of repressor proteins are present in the cells inhibiting the expression of GFP. This nicely tuneable pRha can be used for rhamnose-mediated characterization

Next we characterized pCI/Lac with our construct pCI/Lac gfp using the rhamnose-mediated system. First the constitutive promoter strength of pCI/Lac is determined using the results of the fluorescence measurements and compared to pTet. The RFU values of E. coli NEB5α strains containing pCI/Lac gfp and pTet gfp are shown in Figure 3. As can be seen in this figure, pTet is a stronger promoter than pCI/Lac. Using the results shown in Figure 3 the RPU value of pCI/Lac is determined using the known value of pTet as stated by Kelly et al. 2009 [1]. Data from time point 8.25h was chosen for calculation. Compared to the pTet RPU value 1.5, pCI/Lac has a RPU of 1.0.

We further characterized the promoter pCI/Lac at different rhamnose concentrations. Figure 4 shows that a higher concentration of rhamnose gives a lower RFU value. This means that the GFP expression is repressed by the repressor protein produced under the rhamnose promoter. As shown in Figure 2 a low concentration of rhamnose (0.001%) does not have any substantial effect on the expression rate of the protein. This is also visible in Figure 4 in which for both CIλ (A) and LacI (B) 0.001% rhamnose does not show a lower fluorescence compared to 0% rhamnose. We expected the same RFU values for cultures grown in medium with 0.2% glucose and 0% rhamnose, since glucose functions as a repressor for pRha. Though, cultures grown in medium with glucose show lower RFU values in both graphs in Figure 4. This can be explained by the higher growth rate of the cells in glucose containing medium, since the RFU value is OD dependant (notebook). In Figure 2 0.01% rhamnose shows a higher RFU compared to the culture grown on 0% rhamnose, which indicates that pRha is active at that concentration at that time point. However, at the concentration of 0.01% rhamnose both CIλ (A) and LacI (B) do not show any substantial repression. We can conclude that the concentration repressors produced at the promoter strength of pRha in 0.01% rhamnose is not high enough to perform repression of the promoter pCI/Lac. Though, cultures grown in 0.05% and 0.2% rhamnose show a lower RFU value for both constructs as the RFU in Figure 4 increases in higher rhamnose concentrations. We can conclude that repression of pCI/Lac is stronger in higher concentrations rhamnose, since higher concentrations rhamnose lead to a higher concentration of repressor proteins.

Conclusion

In the last month we made characterisation plasmids to characterize the registry promoters pCI/Lac and pCI/Tet the new set of promoters. Unfortunataly, due to a lack of time we were not able test them all using our rhamnose-mediated method. By the succesfull characterization of the double repressible pCI/Lac we gave a proof of principle for characterizing non-chemically inducible promoters. In future work we could finish characterization of more promoters to get more experience with this new characterization method for further optimization of our protocol. For instance, different rhamnose concentration could be used. When fully developed, this could become a standard method for characterization of repressible promoters.

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References

1. Kelly, J.R., et al., Measuring the activity of BioBrick promoters using an in vivo reference standard. J Biol Eng, 2009. 3: p. 4.
2. Wegerer, A., T. Sun, and J. Altenbuchner, Optimization of an E. coli L-rhamnose-inducible expression vector: test of various genetic module combinations. BMC Biotechnol, 2008. 8: p. 2.