Details of our promoter: pBAD Strong

We decided to utilise the pBAD strong promoter (K206000), which was created by the British Columbia iGEM 2009 team as a mutagenized form of the naturally occurring pBAD promoter. The modelling was based upon and used references pertaining to the pBAD promoter, also known as the 'ara operon'. pBAD and pBAD strong are both arabinose-inducible promoters.

In the absence and presence of arabinose, the pBAD promoter acts as a repressor and an inducer respectively. The gene products of pBAD in Escherichia coli allow the cells to take up and catabolize L-arabinose, a five-carbon sugar. pBAD’s very basic structure is depicted below:

Figure 1: A basic structural outline of the pBAD and, adjacent, AraC protein, in the absence of arabinose. (Schleif, 2011)

The three enzymes that comprise the pBAD promoter cause the catabolism of the sugar arabinose as follows:

araA – arabinose isomerase which converts arabinose to ribulose

araB – ribulokinase which phosphorylates ribulose

araD – ribulose-5-phosphate epimerase which converts ribulose-5-phosphate which can then be metabolised via the pentose phosphate pathway (Patel, GU)

Adjacent to the 3 pBAD structural genes is the AraC regulatory gene. The dimeric (the compound comprises of two structurally similar subunits called monomers which together make a dimer) AraC protein actively represses transcription as well as the synthesis of the pBAD genes; this occurs when arabinose is not present in the environment. The AraC protein binds to the half sites araO₂ and ara I₁ which creates a loop within the DNA, thus blocking the RNA polymerase from binding to the pC and pBAD promoters. This can be seen in Figure 2, where it’s possible to comprehend the position of the relative half-sites.

Figure 2: The red circles in the AraC pockets denote the inducer, L-arabinose. (Schleif, 2003)

When arabinose is present, it binds to the AraC protein and this destabilises the AraC protein binding to the araI₁-O₂ half-site looped complex, but stabilizes binding to the adjacent half-sites araI₁ and araI₂, which are upstream of the pBAD promoter. This then ‘straightens’ the DNA loop, (Carra and Schleif, 1993) allowing the activation of the transcription of pBAD (Schleif, 2011).

Creating a model

The modelling was established using the theoretical equations used in St Andrews 2010 iGEM team which can be found in their wiki under the subheading “Typical ODE Elements”. Our system is a series of five ordinary differential equations which attempt to mathematically describe the very basic functions in our biology, they are as follows:

List of Equations

Notation Table

Programs Used

We considered coding our own basic iterative method but consequently decided that there may be too many complications and bugs to worry about so opted to perform our modelling in the MATLAB environment. We also opted for the same strategy that last year’s St Andrews team adopted, namely the 4th order Runge-Kutta solver. This solver is inbuilt to the MATLAB environment, and is stated as ode45.

Parameters Used

The various parameters were researched throughout previous iGEM team’s findings, several journals and numerous articles. The initial relevant findings and their sources are given below:

Referring back to the equations, it can be seen that we had difficulties in locating five of the parameters within any literature. These particular constants and constraints were attempted, via parameter sensitivity analysis, at being established. We used ranges for these elusive parameters to find optimal values.

Parameter Ranges

These rates were estimated from a variety of sources. The optimal extracellular arabinose concentration is actually assumed to be around 1x10-6M, as if the extracellular arabinose concentration exceeds 1x10-7M then it allows the induction of the AraC promoter [AraC protein, regulation of the L-arabinose operon in E.coli….]and according to the Standard Registry of Biological Parts [1], the ‘switching point’ for arabinose in pBAD is 3.14x10-6M.

Initial Concentrations

Results

There was a significant amount of output from the model as we had a considerable volume of parameters to compare against. 5 subplots were created from the model, which were in reference to the time evolution of the concentrations of intracellular arabinose, arabinose-AraC complex, AraC protein, mRNA coding and protegrin-1 respectively. For the production of the protegrin-1, mainly encased in equation (1.5), the parameters are unknown so there was a great deal of variation in the final graph produced in the output. Below are two selected outputs from the model which depict the different behaviour and the possible instability of the protegrin-1 production.

Complications

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Conclusions

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References:

Patel, Bharat. "Regulation of Gene Expression in Prokaryotes." Lecture 4. Griffiths University. Link to paper.

Schleif, Robert. "AraC Protein: A Love-Hate Relationship." BioEssays, Vol. 25, pg. 274-282. Published 2003. Link to paper.

Schleif, Robert. The Johns Hopkins University. Last updated August 2011. Link to paper.

Carra, John and Schleif, Robert. "Variation of half-site organization and DNA looping by AraC protein." The EMBO Journal, Vol. 12, pg. 35-44. Published 1993. Link to paper.