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Team:Consort Alberta/project
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Our Project
Comparison Final Result Prototyping
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 two 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.
ECOS which 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, causing mutations and is carcinogenic as a result.
In order to create a visual representation of our output, we turned to mathematical modelling. Using constants and values from online research 1&2 we created five equations to represent the action of our biobrick producing the protein XylR, binding with xylene and relationship to the output of our respective proteins. The following graph shows the estimated overall output of our biobrick. 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.
***GRAPHHHHHHH
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. J23100TC represents the activity of our promoter. KRNAdeg 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. XylRi and XylRa refer to the concentrations of the inactive and active forms of XylR. KJ23100 is the production rate of the promoter BBa_J23100, rXylR is the oligomerization constant of XylR, rR,XylR is the dissociation constant of inactive XylR and xyl in the total concentration of xylene. αXylRi 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. XylRi and XylRa refer to the concentrations of the inactive and active forms of XylR. rXylR is the oligomerization constant of XylR, rR,XylR is the dissociation constant of inactive XylR and xyl in the total concentration of xylene. αXylRi 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. PuTC represents the activity of the Pu promoter or the output of protein. αpu is the deactivation rate of the Pu promoter which we are assuming is alike the Ps promoter due to their similar function and activation rates. KXylRa is the repression coefficients of the Pu due to binding and npr,i and npr,a are the hill coefficients of the Pu promoter. β0 and βPS are the basal expression of the Pu promoter.
E. coli Plasmid Design: The first portion of our plasmid is essential to the way our xylene detector is going to work because it will be responsible for constantly producing our transcriptional regulator XylR which recognizes the chemical m-xylene and undergoes a conformational change which then allows it to bind to, and positively regulate the pu promoter at a 4 XylR to 2 xylene ratio starting the production of our reporter protein. 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.
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: a) E0040 - GFP wild-type, will glow when exposed to UV light. b) K592009 - amilCFP, This chromoprotein from the coral Acropora millepora, naturally exhibits strong color when expressed. c) 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.
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.
PROTOTYPE #2: We answered our problem by having our ECOS impregnated into alginate and then made into small spheres so that we can just add them into a heated solution/sample to get our results. In a container, we combine our soil sample and our alginate beads, mix them around, and then sift out the beads. The beads produce our indicator protein from the contamination levels of the soil. This design was inspired by the Peking 2013 Team that used alginate encapsulation beads. Their reasoning and justification for using alginate beads stood out to us, as they stated that it was stable and inexpensive and easy to shape and manipulate. Upon further research into the use of these beads, we discovered that this prototype could be more efficient to use then our first prototype that required a few different pieces to the system. This revised prototype would also be easier for the average oil worker to use compared to the larger prototype.
References: 1: Calgary 2012 ECHEM Values, 2: Wiley Value References
