Engineering Success
Our Project’s Little story
For our team to design and produce a working Diagnostic platform, our engineering journey had to pass through many design-build-test-learn & Improve cycles to get the platform to be able to:-
1) Have Optimal Function & Diagnostic accuracy
2) Be as cost efficient as possible
3) Serve as a modular diagnostic Platform that can be applied for multiple diseases
4) Add a therapeutic element to our diagnostic kit
This Journey started early with the research & brainstorming stage. We tried to tackle the national problem of inborn errors of metabolism as we have found out that our country faces an absolute catastrophe with one of the highest numbers of children affected with Inborn error of metabolisms (IEMs).
Iteration 1
Our First approach to tackle this national problem was to try to use synthetic biology to design a cost-efficient and reliable screening kit for the public and that’s where our early research led us to try and work on Microfluidic chips as our first iteration to design our diagnostic kit. We found through literature that the Microfluidics-based immunoassay system is a promising example for the point-of care diagnostics being affordable, sensitive, rapid, and robust. Thus providing an easy way to rapidly detect diseases at extremely low cost and short time.
Our First iteration to build such a kit made us contact experts in the field of Microfluidics & Engineers which helped us thoroughly in designing our preliminary plan and model for the chip. We designed that model to solve the problems that we might encounter in the lab while using this system. Then began our search to contact labs all over the country that might be able to sustain our needs by providing us the required equipment and lab setting for designing that kit.
After Contacting several Experts and laboratories we faced many problems such as:
1) Lack of needed tools and equipment needed for the production and designing of the chip
2) Absence of clear rooms in the country that are required for the fabrication and scalable production of microfluidic chips. Because microfluidic chips have very narrow pores and could easily be clogged by tiny dust particles making the whole system fail.
3) Unsustainability of long term production of the chip even if it was to be ordered from across the country
4) Hard Use of the microfluidic chip that contradicted our need of the chip to become a screening tool with wide use in the country, with only proper experts being able to work on it.
5) Presence of many faults that can happen to the chip & the need for the test to be conducted exclusively under certain conditions.
Our search provided us with the knowledge to go through and re-research a more Viable solution that would go hand in hand with what we need to provide a sustainable and cost efficient solution and led us to disregard the use of microfluidic chips as a whole.
After Finding out that the use of microfluidic chips is something to be considered problematic we needed to start searching for a new solution to be used for designing the kit. We went on a visit to the nanocentre in AUC (American University in Cairo) to search for other lab facilities that could help us find another application for our diagnostic system. We met there Dr. Reda Abdelbaset who was of great help to us and told us about another system similar to the Microfluidic chips, it is called Printed Circuit Boards (PCB). But we faced the same problems because these kinds of chips are not available in Egypt and had to be purchased and imported to be able to use it for our system.
After contacting several doctors such as Dr. Eran Andrechek, associate professor in physiology. and telling him about our project, we had many discussions over some aspects regarding our project and what we might need to be able to practically apply our idea in a simple way. He recommended the use of Lateral flow assays as it would be much cheaper, more affordable and if executed properly would enable us to implement our design in a very easy and effective way.
In Addition to that Dr. Farid AboElelaa Associate Professor of Biophysics in Zewail City of science and technology. suggested the use of aptamers, which are oligonucleotide molecules that can selectively bind to a specific target at a very small scale. Aptamers can be more specific antibodies as a biological recognition element that can easily detect small quantities of analyte in a given sample.
We started conducting our own research on lateral flow assays. We even estimated the prices to conduct similar tests & their effectiveness with what we had in mind as well as started researching aptamers in comparison to the use of antibodies.
We learned that lateral flow assays best possible candidate and would be both cost effective and cheap to produce, as well as be easier for people to use at home as point of care diagnostic, and we were able find the lab material for it in our country also we found that aptamers would be a great benefit to us and our diagnostic kit as it would add much more to us regarding specificity and sensitivity to the intended targets
In the early phases of designing our project we needed to know how to make our kit as user friendly as possible so we thought first about using fluorescence as our mainstay for the detection method so it would be as simple as possible for the people to understand and work with the test results as we were considering our people while working. We face another problem: how the cell detects substances outside it.
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In working then to build a solution we started studying about fluorescence and different types of it and how we could build a circuit that can incorporate fluorescence, but we were faced with a brick wall as it was found out that fluoroscopy would be very expensive and hard to reach modality in our country so we started asking professionals about alternatives and we had meetings with Dr. Eran Andrechek who suggested that the use of colorimetry would be a feasible alternative as a detection system in general and would serve our goal for easy test readability & understanding as well as be much more less in cost if designed and built properly
Test & Research
We started testing his hypothesis and researching literature to find a method that in addition to the easy readability of colorimetry would maybe help our kit to be not only be a qualitative method of detection (Positive & Negative) but also have semi quantitative factor to maybe actually give the user a hint of the severity of his current disease and we found that gold nanoparticles (AuNP) would be the key to as we found out induced aggregation of the particles can cause different color changes that can actually by interpreted according to the amount of present detected biomarker.
So we learned that colorimetry induced using AuNP aggregation would be a the best fit for our project and long-term goals of designing a point of care diagnostic kit
Iteration 4
In the process of determining how we are going to make a diagnostic kit for PKU we needed to start first researching what are the most relevant biomarkers that would actually indicate the presence of a disease and avoid using insensitive biomarkers for our diagnostic device. Accordingly we had to filter from a panel of possible target molecules.
So in order to do that we had meetings with pediatricians and collected more information about this disease as well as conducting meetings with multiple experts in the field such as Dr. Nashwa ElKhazrage.
After doing our own research & contact with her and multiple meetings which helped us determine that the best marker to be detected would be phenylalanine
We decided that the biomarker to be targeted would finally be phenylalanine because it would be the most sensitive to PKU as well as easy to detect; however this posed another problem to us.
Although using phenylalanine as a biomarker would be a good fit in our project and a sensitive indicator of the disease in excess and represent enzymatic deficiency, It had normal blood levels so our kit needed to not only be able to detect the biomarker but to distinguish between its mere presence and excess of it.
As Always we started our work by regrouping, researching and contact with experts to try and determine how we could design a kit that is able to detect only the excess of the biomarker that’s when we came with a novel idea of making our Lateral flow assay containing a line that would consume all the phenylalanine that can be present in normal humans and only for excess of phenylalanine to be able to interact with the second line of our LFT indicating presence of disease the lines of which we call the The Jury & Fugitive lines.
In order to test and validate our consumption line we started by preparing 5 tubes of different concentrations of aptamers after that we started introducing a constant amount of phenylalanine which is considered the upper limit of the normal range in humans which is 20 mg/dl after that we witnessed an increase of the color change in all tubes except for tube no. 5 which matched the color of tube no. 4 indicating that the concentration of aptamers no. 4 was enough to consume all the normal levels of phenylalanine.
By doing our experimental test we were able to recognize the concentration needed in order to prepare our consumption line to be able to accurately reduce false positive results and enhance the design of our Lateral flow assay.
Iteration 6
Our protocol first was to start by preparing 2 DNA fragments that are functionalized with Gold Nanoparticles that form purple aggregate as long as they are annealed to each other. Nanoparticles 1 and 2 are functionalized with two different DNA molecules through thiol-gold chemistry.
The bonds with the initial part of the aptamer are stable at room temperature. However, they are more likely to disrupt once the aptamer changes to its secondary structure upon recognizing the biomarker of interest.
Once the aptamer binds to the analyte, it detaches from the second DNA fragment and surrounds the biomarker, leaving the second DNA fragment paired only with the Penta-nucleotide sequence. which are very weak, unstable and prone to disintegration in that case. As a result, the two nanoparticles functionalized with dna molecules are disassembled and separated from each other, changing the color of the gold nanoparticles from purple to red.
Build:
After starting to prepare our protocol we found a problem with it as we intended for it to be used as a consumption line on lateral flow assay however we found that It was too hard to actually stabilize the aptamers on the lateral flow assay as we have found out that the protocol would leave the aptamers unstable at room temperature.
So as always we started researching and redesigning our protocol to find a protocol that would give us accurate results and sensitive binding as well as be able to stay stable under room temperature that’s when we came across the duplex dissociation mechanism which is a protocol that would allow our aptamers to maintain a fast paced detection and high sensitivity for small molecule, this method works by the ability of nucleic acids to form Watson-Crick-Franklin base pairing, making the end result being a three dimensional configuration that is formed of the desired target analyte with high sensitivity and specificity which uses a complementary strand attached to the aptamer which upon introduction with a ligand would displaced from its place due to conformational changes. This induces signal transduction by the complementary strand’s separation.
This protocol has many advantages over the previous one, because it was more applicable on lateral flow assay. As we can easily immobilize the control probe DNA using biotin labeling technique as well as being much easier to integrate with the consumption line using streptavidin-biotin DNA conjugates technique.
We prepared for the validation of our protocol by preparing test tubes with the aptamers using the new protocol by preparation of different concentrations of aptamers in test tubes followed by introduction of phenylalanine samples with the same concentration which was predetermined using spectrophotometry.
(Edit:Sowar Test Tubes & Revision of testing methodology)
Our results indicated colorimetric changes in the test tubes upon introduction of phenylalanine with different color grades indicating appropriate and successful binding of phenylalanine with the aptamers designed under the new protocol which would be stable under room temperature.
Iteration 7
After receiving our order from IDT and finishing our drylab work and theoretical preparation we made designs using Synthetic Biology Open Language (SBOL) of our circuit and started preparing our protocols needed to be able to work in the lab.
Build:
In order to start making our circuit into an actual device to be tested and used the first step to do was first doing resuspension of the parts followed by digestion and ligation of it and preparation of it to be transformed
However to go through the next step we needed to characterize our parts and to first use western plotting and the use of our gel electrophoresis to characterize and validate our parts and the process of digestion and proper ligation of these parts using our knowledge of their molecular weight and the DNA ladder used as a reference and control. So we can go through the steps of transformation.
However after doing that we have found that the gel showed weak bands in both the digested and ligated parts indicating that there is weak concentration of the parts
We learned that we had weaker concentrations than what would be needed and that we need to use PCR to amplify our parts in order to get stronger bands and be able to correctly characterize and verify our parts.
In our First attempts and designs to use and introduce colorimetry and to design our whole cell biosensor that would be able to detect the excess of phenylalanine from the consumption line we intended to use CRPG outside the cell to react with beta-galactosidase enzyme as it converts the CPRG substrate which is yellow-orange into a red chromophore called chlorophenol red turning the final solution into dark red color.
However after the construction of the idea and design we were faced by the lack of CPRG in our country as it there were no sellers for it in here so we needed to start researching and redesigning
After searching for an alternative to CRPG we found an organic compound called X-gal which is made from galactose bound to a substituted indole
To test X-Gal in the lab we introduced it to E.Coli that was pre-made using transformation techniques and prepared an agar to try and see for ourselves how it would look like in the lab as well as be able to use the result as a control for future lab work as this would be considered as the control and normal expected outcome of phenylalanine detection and its forthcoming expression of b-galactosidase.
After doing our experiment & studying the compound we learned that X-gal would even be a better solution to our problem and even better than our early suggestion of cprg because it gives blue color which is more appropriate for color blinded people making our future device more friendly for those with disabilities.
We designed our project to sense phenylalanine using a whole cell-based biosensor that works by our genetic circuit, which involves a phenylalanine-sensitive regulator called TyrR to express b-galactosidase in the presence of phenylalanine for it to react with X-gal and produce blue color .
We started building this circuit by transforming it into E.coli using transformation techniques and started our process of assessment of color production in the case of phenylalanine introduction.
After introduction of phenylalanine to the circuit we found that there were almost no color change that was presented or happened so we started search in for the cause of this and we found that there were a flaw in design because phenylalanine is extracellular and TyrR is intracellular so we were unable to express Phe to TyrR
To solve this problem we added another circuit to produce the permease enzyme which makes the cell membrane permeable to phenylalanine
After retesting again with the addition of permease expression we found that there were still weak colors unlike what was seen in the control with the X-gal and b-galactosidase was due to the low concentration of beta-galactosidase outside the cell
So we started searching for a signal amplifier to add to our circuit that would be able to increase the color in the case of detection of phenylalanine and after research and meeting with doctors we decided to ligate KP-SP to the Lacz promoter to be able to secrete beta-galactosidase extracellularly.
We started Transforming our new design again into E.coli in order to determine whether the addition of Kp-Sp which is a secreted signal peptide and whether it would be able to carry b-galactosidase extracellularly by its tagging to lacz alpha
And to test our new circuit we used comparative methods to compare between the previously made agar with another with the same concentration of phenylalanine with the only change about it being the addition of Kp-Sp tagging to the second plasmid.
We learned that the addition of Kp-Sp made a remarkable change in the color concentration and the amplification of the color signal and so this modification in our circuit was finalized
When we primarily designed our first design for the therapeutic system for PKU the design would work by the detection of phenylalanine and tyrosine levels and then further expression and regulation of phenyl hydroxylase enzymes in accordance to the levels of the selected biomarkers above.
However we knew In our design of the therapeutic circuit that the primary biomarker that would indicate the success of our therapeutic circuit is the expression of phenylalanine hydroxylase as we needed to to monitor degrees of PAH synthesis within the cells so we planned to use PAH elisa kit. .
Build & Solve
Prior to the preparation of our therapeutic circuit and beginning to work on the protocol for transformation we have found out that the process to extract the media and seperate it from the cells to repeatedly be used on elisa kit for verification would be time and material consuming as well as it can be something that would increase future costs of manufacture of the product
So we started searching for an alternative to the usage of an external kit for validation and we started thinking of methods that are inherently built in the circuit and to find a solution that would be able for our circuit to be tested under any condition so we thought of incorporating one of our previously used parts which is the Lacz alpha that would produce b-galactosidase and by using P2A as a linker between it and PAH, so production of color in the media in presence of X-Gal would represent PAH expression.
In order to test whether our system can effectively produce PAH and LacZ alpha as a reporter gene simultaneously, as well as whether attaching LacZ alpha to PAH will decrease the amounts of PAH expression, we transformed the newly designed circuit into E.coli and used phenylalanine samples with concentrations predetermined using spectrophotometery to induce the expression of phenylalanine hydroxylase.
After conducting our experiment we have found out that there have been color expression indicating the release of phenylalanine hydroxylase from the circuit therefore indicating success of the circuit’s design and ability to express its intended target outcome.
In order to tune our circuit’s activity and prevent over-expression of PAH, we had to integrate a regulatory system with our circuit, so after research we decided to utilize CRISPR system which originally was found in the genome of bacteria as an immune system against bacteriophages, and to incorporate this system into our design we added an additional circuit which would consist of two parts.
The
first part would be a guide RNA which would have complementary sequence to the
target sequence with high affinity for it and this complementary sequence would
be placed upstream of the PAH codon sequence that needs editing, and second
part would be, Cas9, an RNA-guided protein that edits genes by cleaving the two
strands of the target gene simultaneously through its nuclease activity after
recognizing PAM sequence tied to the PAH gene. This will terminate the
expression of PAH to sustain its concentration within the normal level.
So we started designing our new circuit that
incorporated the crispr system and we started transforming this circuit with
the previously made circuit to E.coli to test it. To
assess how well our circuit could regulate and tune the level of PAH expression
under various conditions, after we transferred it to E. coli. We discovered
that PAH expression is reduced when phenylalanine levels are low, but even when
we increased the concentration of phenylalanine, the levels of PAH were still
relatively low to phenylalanine concentration. As a result, we performed DNA
sequencing to identify the cause of PAH non-functionality. Learn
& Improve DNA analysis
revealed frameshift mutations in the PAH coding sequence and an off-targeting
effect due to interactions with other coding sequences, so we had to evolve our
regulatory system to operate at the mRNA level rather than genomic level so
that that the crispr system and safety mechanism would only be modulatory and
repressive of the expression of PAH rather than complete termination of the
function of the circuit as well as prevent mutations and accidentally knock
down other genes containing the PAM sequence. That’s why we replaced Cas9 with
dCas12g, which acts as a post-transcriptional modifier by targeting the mRNA of
PAH and restricting its translation. 
