Team:Consort Alberta/project

Our Project

   Introduction    The Science   Comparison 

Mathematical Modelling    Prototyping

Introduction...

     ECOS is a biological sensor theorized to detect aromatic hydrocarbons by producing an indicator protein when exposed to xylene. We chose xylene as our trigger because it is very well correlated with the presence of other more dangerous compounds such as benzene and benzene derivatives. The danger of this class of compounds is in the fact that they can intercalate into DNA and cause mutations. For this season our team is working on expanding and perfecting our project. Our goal is to create a biobrick and a working prototype that will detect levels of xylene, which is linked directly to carcinogens found in petroleum products such as benzene and its derivatives. Our biobrick will create different amounts of fluorescent protein in the presence of xylene bound to xylR. The economy in our rural community is based largely on agriculture and oil and gas industries. Oil spills have detrimental effects on the environment, economy, and general health. This year we will be testing out three different indicators to allow options concerning scale and intensity of colour change. In future years our project will allow early identification of contamination will facilitate rapid clean-up and minimize health risks to members of our community and to the consumers who rely on the food we produce.

The Science...

    Our resulting biobrick consists of two main portions. The first portion of our plasmid will be responsible for producing the protein XylR. This is essential because the bonding of m-xylene undergoes a conformational change when bonded to XylR which then allows it to bind to, and positively regulate the Pu promoter. At a 4:2 XylR to Xylene ratio the bonding starts the production of our reporter protein. The second portion of our plasmid expresses the indicator protein when in the presence of bonded XylR and m-xylene and essentially allow us to know if m-xylene is present in our soil sample or not. The structure of this biobrick is shown below.

     Parts:

1. J23100 - which is a constituent promoter.
2. B0034 - the RBS for our XylR gene.
3. I723017 - the XylR coding region which encodes for the transcriptional regulator XylR protein.
4. B0015 - the double stop codon for this sequence.
5. I723020 - This is the Pu promoter.
6. B0030 - RBS.1 strong.
7. Our indicators which we will be testing so that we can see which indicator for our project will be the best:
1. E0040 - GFP wild-type, will glow when exposed to UV light.
2. K592009 - amilCFP, This chromoprotein from the coral Acropora millepora, naturally exhibits strong color when expressed.
3. I732005 - lacZ, Beta-galactosidase cleaves X-gal and ONPG into colorful products.
8. B0015 - lastly another double stop codon.
9. pSB1C3 - Our plasmid backbone which includes the resistance towards the antibiotic, chloramphenicol.

Comparison...

     Where the comparison portion comes into play is the second part, which will be chosen from any one of three indicators: amilCP which when produced expresses a strong blue color, GFP when under a UV light source fluoresces green or, LacZ which when a substrate is added it cleaves it into strongly colored blue products. We will be looking at which indicator functions with the most efficiency and produces results of the highest clarity.

Mathematical Modelling...

     In order to create a visual representation of our output, we turned to mathematical modelling. We created a modification of a model done by Koutinas, M., et al.^[1] which was modelling the expression of the Ps promoter. The Ps promoter is the natural promoter found with the xylR gene. Due to similarities between the Ps and Pu promoters, we assumed the deactivation rate of the two components were alike. For remaining values, we simply replaced the values of the Ps promoter with known values of the Pu promoter. ^[2,3] In the end, we created five equations to represent the action of our biobrick. Our model shows our biobrick producing the protein XylR, binding with xylene and the relationship to the output of our respective proteins. Each of our formulas are constructed as a rate in which a concentration or output is given of a specific substance in respect to time- otherwise known as a derivative. We will now expand upon the equations we modified and used.

      The first equation above represents the translation of RNA into the protein XylR. RNA is the representation of the rate of DNA being translated into the protein XylR. P represents the number of plasmids inside a cell. J23100_TC represents the activity of our promoter. t_a is the transcription rate of the xylR into RNA, and K_RNAdeg represents the degradation rate in the E.Coli.

      The second and third equations represent the inactive and active forms of XylR- or the concentration of XylR that has not bonded to xylene and the concentration of XylR that has bonded to xylene. XylR_i and XylR_a refer to the concentrations of the inactive and active forms of XylR. RNA is the substitution of our first equation. t_r is the translation rate of our RNA into XylR. r_XylR is the oligomerization constant of XylR, r_R,XylR is the dissociation constant of inactive XylR and xyl in the total concentration of xylene. α_XylRi accounts for degradation and dilution because of cellular volume increase. Xylene represents the concentration of xylene present.

      The fourth equation represents the concentration of xylene present. XylR_i and XylR_a refer to the concentrations of the inactive and active forms of XylR. r_XylR is the oligomerization constant of XylR, r_R,XylR is the dissociation constant of inactive XylR and xyl in the total concentration of xylene. α_XylRi accounts for degradation and dilution because of cellular volume increase. Xylene represents the concentration of xylene present. We multiplied by seconds per hour to get a domain more realistic to our needs.

      The fifth equation represents the output of the pu promoter. Pu_TC represents the activity of the Pu promoter or the output of protein. α_pu is the deactivation rate of the Pu promoter. K_XylRa is the repression coefficients of the Pu due to binding and n_pr,i and n_pr,a are the hill coefficients of the Pu promoter. β₀ and β_PS are the basal and maximal expression respectively of the Pu promoter.

      In order to create an output, we used the program Scilab. By inputting these equations into code we were able to have the program calculate our estimates and graph the results. In the end our output was around 5.5 mPoPS. This is a logical output and tells us our biobrick will successfully over express the protein. Below is a copy of our code that we had used in our program. The resulting graph is the output of our Pu promoter.

Prototyping...

PROTOTYPE #1: We designed our first prototype so that it has a positive pressure source which creates a current for our xylene to travel from our sample, which is in a heated container so that the xylene is vapourized, to our ECOS container in which we bubble it through the E. Coli culture to get results. This was a plausible idea, as it would require fairly non-expensive materials and could be maintained by the average business person. However, we thought it could be more efficient in it's design, and so we kept looking for ideas. Our biggest problem with this was the fact that it is a relatively complex prototype. So we challenged ourselves to try and create a simpler prototype.