Team:Thessaly/Experiments
EXPERIMENTS.
The workflow of our experiments was based mainly on the design of our project, which consists of 3 basic parts. The amplification of the biomarker sequence, followed by in vitro transcription and translation, resulting to a color-changing reaction. In this page you can find the protocols used during our experiments, our detailed experimental design, and the Lab Book of our project.
Protocols
For an experiment to be rightly executed, some basic protocols must be followed. Here is the list and the detailed description of the protocols we used, in order to get through the experiments. To download our protocols click in the boxes below.
Experimental Design
IS6110 preparation
The first step of our experiments was to prepare our biomarker, the IS6110 gene fragment. We first had to order the sequence of the IS6110 fragment from IDT (Integrated DNA Technologies) and after diluting it to the desired concentration, to amplify it with a set of primers that anneal at the edge of our DNA fragment. This step was necessary in order to amplify our sequence and keep it stored so that it can be used at later steps as a template for our experiments. It is important to note that our fragment was ordered without the prefix and suffix sequences, thus, in order to make it RFC10 compatible, we added these sequences as overhangs in the abovementioned primers. After obtaining this sequence in many copies, we had to quantify it and later create a series of dilutions of our fragment so that they could be used later as a template to test the efficiency of our amplification method (RPA).
Figure 1. Vector map of pSB1C3 containing the IS6110 gene fragment (987bp)
DNA Amplification using Recombinase Polymerase Amplification (RPA) and PCR
Our first step was to check if our chosen biomarker (IS6110 element) can be amplified, first with a PCR reaction and then with the Recombinase Polymerase Amplification (RPA), which is an isothermal amplification method. In order to determine the reaction’s optimal conditions for the selected DNA template, we conducted a series of experiments. Ultimately, we were able to show that the amplification of our DNA biomarker can be achieved using both PCR and RPA.[1]
Briefly, we examined the amplification performance both for the PCR and the RPA reaction when altering the following reaction conditions, one at a time, thus standardizing the methods:
a. Amplified product length
To test the reaction’s efficiency when amplifying different product sizes, we designed three sets of primers on our DNA template, corresponding to amplicon length of 80, 250 and 500 bp respectively. The reverse primer is the same in every set to ensure the similarity between the sets, in order to clarify if product length plays a role in amplification performance.[1]
b. Annealing primer length
Primer quality is a critical component of these reactions and especially for the RPA reaction. Even differences in a few nucleotides can increase the specificity of the reaction a lot. To address that, we designed three different reverse primers for our DNA template, each one with a different annealing length. Moreover, we sought to cover the range of optimal primer length according to RPA’s manual, thus the 3 oligos were 32, 36 and 39 bp in length respectively.
c. Addition of 5’ end overhangs in the primer sequences
In order to uncouple downstream signaling from the specific sequence of our DNA template, whilst creating a modular and universal tool, we designed primers with additional sequences at their 5’ end. With a few publications on this matter, we sought to test reaction performance in presence of universal overhanging sequences.
Note: To see a detailed description of our in silico primer design, click here.
d. Incorporation of PCR additives
Even though RPA is a great method to amplify and detect DNA in a very fast and cheap way, some intrinsic issues lie within its molecular mechanism. This results in frequent primer-dimer formation and presence of several RPA artifacts, along with the amplified sequence. To eliminate those, we tested some common PCR additives that are known to enhance a PCR reaction’s specificity, both for the PCR and the RPA reactions. Moreover, since Mycobacterium tuberculosis and thus our biomarker has a high GC content, those additives can help alleviate secondary structures in the template. To be precise, we tested the PCR performance using DMSO (Dimethyl Sulfoxide)Molecular grade DMSO is a PCR additive which is commonly used for the amplification of GC rich templates. It enhances the reactions performance by facilitating the specific annealing of the primers to the template and thus, reducing non-specific amplification at a final concentration of 5% and the RPA performance, also with the addition of DMSO (2.5%, 5%, 10%). While DMSO improved the PCR performance significally, it seems to inhibit the reaction of RPA, and that is maybe the reason for the lack of literature on the matter[2].
To further standardize the RPA method, which is the amplification method we chose for our project design, we conducted some more experiments to check different conditions for our method and establish which of those are the optimal ones.
e. Incubation temperature (25℃, 37℃, 39℃, 42℃, 50℃)
Another major requirement is the conduction of the assay without using specialized equipment. Thus, heating in a specific temperature can decrease the overall impact of the test. RPA has a recommended incubation temperature of 39℃ , so in order to explore the limits of that, we tested reactions at 25 (RT), 37℃, 39℃, 42℃ and 50℃, showing that while the optimal range is between 37-42℃, the reaction can be conducted in either higher or lower temperatures.
f. Template Concentration
One of the most critical aspects of a diagnostic test is the assay’s sensitivity and thus the Limit of Detection (LoD). In order to assess the LoD of RPA with our biomarker, we prepared serial dilutions of the template, down to 1 copy per μl (corresponding 10-9ng/μl). Furthermore, we conducted RPA reactions successfully with 1, 10, 102 , 103, 104, 105, 106, 107, 108 copies of our biomarker per reaction respectively.
g. Incubation time (3, 5, 7, 10, 15, 20 min)
A requirement of Point of Care (POC) tests is that they can produce a reliable signal in-field, in a small fraction of time. Thus, we assessed RPA performance in decreasing incubation time. Furthermore, starting from the recommended incubation time of 20 minutes, we decreased it to 15, 10, 7, 5 and 3 minutes respectively, managing to amplify even a single copy in a 5-minute reaction.
Combining the knowledge gained from the standardization process, we performed the final optimized RPA reactions for our MTB biomarker, in a modified protocol where incubation time is 5 minutes, the temperature is 42℃, while being able to detect even a single copy of the template per reaction.
To further enhance our proof of concept and make the conditions of our experiments seem more realistic, we decided to test our diagnostic test, simulating the natural fragmentation of our biomarker (IS6110) in patients’ urine and see if we would be able to locate and amplify it. For this reason, we used DNase I to our template DNA (987bp), so that random fragmentation would occur, and after that, we were able to still amplify our biomarker with our designed primer set and in much more realistic conditions.[4,5]
In vitro protein synthesis assay
The second experimental installment of our project was the expression of the protein of interest, through a cell-free system. The goal of this experiment was to prove that the device we have designed for DNA detection can produce a robust colorimetric readout, both visible to the naked eye and measured in a plate-reader spectrophotometer. To achieve that, we conducted several preliminary experiments, to prepare and calibrate the reaction’s conditions, as well as produce the genetic material used in the reactions.
The steps we followed towards our goal are:
In order to have enough genetic material for all our experiments, we spared some time to clone every BioBrick we received into pSB1K3, pSB1C3 of pSB1A3 clining vectors. Below you can find a table with the BioBricks cloned and used in our in vitro experiments (all parts have a T7 promoter BBa_J64997 and T7 terminator BBa_B0015).
Table 1. The BioBrick parts that were generated and used in our in vitro protein synthesis assay
The non-regulated parts serve as a measure to compare the regulation efficiency of the toehold switch.
After the cloning process, we isolated the plasmid containing each part in large concentrations and confirmed their identity via Sanger Sequencing. At that point we had enough genetic material to use in our in vitro experiments downstream.
The next step was to test and calibrate the in vitro protein synthesis reaction, due to the lack of expertise in such reactions here in our Department. We used the commercial kit PURExpress from NEB (New England Biolabs) to express the proteins of interest in vitro. Even though there is a detailed protocol available, we optimized its parameters to suit our needs. Moreover, we compared protein expression through an enzymatic assay, between the regulated and non-regulated genes. The conditions we altered are:
a. The volume of the reaction reduced to 7 ul from 25 ul
b. Incubation time (1, 2 and 3 hours)
c. Abundance of trigger sequence in the reaction (75, 50, 16, 7, 3, 0.3 nM per reaction)
d. Different reporter genes (eGFP, β-lactamase)
Our reporter gene of interest is β-lactamase, but during the calibration process we tested eGFP as well, which is a robust reporter protein and can be easily detected, both in vivo and in vitro. As a result, we have provided the community with two evidently working BioBricks regulated by an orthogonal toehold switch.
Note: We ensured that β-lactamase's activity is due to the regulated gene only, because PURExpress utilizes only T7 promoters to transcribe and translate coding sequences.
In order to prove that β-lactamase can be expressed in regulation of an orthogonal switch, we performed a batch of reactions. These included both positive (unregulated gene) and negative (regulated gene without the trigger sequence) controls. After the addition of the substrate in each reaction, we performed an established enzymatic assay for the β-lactamase enzyme for 30 minutes.
Combining the two separate proof of concepts into one
After completing the proof of concept for the toehold switch, we wanted to test the complete functionality of our project and prove that these uncoupled and separate reactions (RPA and toehold switch regulated expression on β-lactamase) can perform as a combined reaction. To test this, we had first to perform the RPA reaction as usual and then use the amplified product as the trigger sequence expressed with the PURE express in vitro synthesis kit, which will trigger our toehold switch to open its formation and enable for the β-lactamase enzyme to be expressed and give us a colorimetric (red) signal through the hydrolysis of Nitrocefin.
The workflow was based on the separate proof of concepts and the standardization of the techniques done previously. The RPA reaction was a 5 minutes reaction, while the product was cleaned after the amplification. Afterward, the amplified and cleaned product was used as a template for the PURE in vitro synthesis reaction which was a 3 hours reaction and then a 30-minute enzymatic assay followed to see if our amplified product can regulate our toehold switch. As a control we used both the β-lactamase enzyme not regulated by any toehold switch and the toehold itself without the triggering sequence, to check the levels of its leakage.
We concluded that with the right conditions, our template biomarker (IS6110) can be amplified by the RPA reaction and then, after its in vitro expression, can give the signal to regulate our toehold switch and lead to the expression of the β-lactamase enzyme and, thus, the hydrolysis of Nitrocefin and a vibrant color change. In conclusion, we showed that our proof of concepts can work together as one and give a universal platform for our diagnostic tool.
Testing our new universal toehold switch
Moving forward from the basic proof of concept, we de novo designed a pool of new toehold switches to create a universal tool. After the in silico analysis two of them were selected to be tested. To prove that the freshly designed switches work, we performed a batch of reactions, following the proof of concept reaction conditions. Briefly, 7μl reactions were performed for 3hours in vitro transcription/translation and the nitrocefin substrate was additionally added. The toehold switch concentration was always the same while the trigger’s concentration was the one changing. We showed that one of the new toehold switches can indeed regulate the protein expression of β-lactamase and produce a robust signal.
Figure 2. Vector maps of pSB1C3 containing the trigger sequence (left) and toehold sequence (right) deriving from the Geobacillus kaustophilus
New disease (Hepatitis B Virus)
With our design we aimed to develop a platform/tool that its nature of approach is universal, and simply by changing the primer set, we can be able to target different pathologic agents, as well as other MTB traits, such as antibiotic resistance. For us to be able to prove the universality of our design we had to test its efficiency when targeting something different. We chose to target a small DNA sequence of the Hepatitis B Virus (HBV), thus proving that with our design, it is possible to diagnose any other disease (if targeting its DNA) only by changing the primers’ sequences. We began exploring this different approach by choosing the targeted region. The region we chose is a small fragment of the DNA sequence of HBV (280 bp) that is part of 3 alternate reading frames. Then we had to redesign our already existing primers, changing only the annealing sequences and keeping the overhanging sequences stable. We designed 3 primer sets, based on pre-existing literature while adjusting their length to fit the RPA method standards.[6]
The first step of our experiments was to order the DNA fragment for HBV as well as the designed primers. After that, when the DNA fragment and the primers arrived, we had to create again a series of dilutions so that they could be used as a template to test the efficiency of our amplification method.
Our goal was to test all 3 primer sets, to check if they can amplify our “biomarker” through PCR and Recombinase Polymerase Amplification (RPA). At the same time, we wanted to check which one of these 3 sets amplifies our fragment most efficiently.
We started testing our primer sets first through PCR, using all 3 sets, while using as a template the lowest concentrations we had created. The conditions were the same as those used for our proof-of-concept reactions, while we used DMSO at a final concentration of 5% in all of our PCR reactions.[7,8]
After achieving the amplification with all of the aforementioned primers, and while having a small clue that the 1st set is able to amplify our fragment best, we continued by testing the amplification efficiency with the RPA method, again with the lowest concentrations of DNA template. The conditions we chose for the RPA reactions were exactly the same as used for our proof of concept reactions, which means that the incubation time was 5 minutes while the temperature was 42℃.
Again, we were able to show that the amplification of our DNA biomarker (this time from HBV) can be achieved using both PCR and RPA, and thus proving that we can use our initial design, with small modifications, to target different DNA templates, making it a universal and modular tool, and simply by changing the primer set, we can be able to target different pathologic agents and more.
Lab Book
One of the most crucial step of the lab work, is to report the daily conducted experiments in detail. Below is our team’s lab book describing our experimental routine. To download it click below.
References
1. TwistAmp® DNA Amplification Kits, Assay Design Manual
2. https://www.sigmaaldrich.com/catalog/product/sigma/d8418?lang=en®ion=GR
3.L. Lillis, et al., Factors influencing Recombinase polymerase amplification (RPA) assay outcomes at point of care, Molecular and Cellular Probes (2016) ” ,Infection. https://doi.org/10.1007/s15010-016-0955-2.
4. Labugger, Ines, Jan Heyckendorf, Stefan Dees, Emilia Häussinger, Christian Herzmann, Thomas A Kohl, Elvira Richter, Eric Rivera Milla, and Christoph Lange. 2016. “Detection of Transrenal DNA for the Diagnosis of Pulmonary Tuberculosis and Treatment Monitoring.
5. Karayiannis, P, D M Novick, A S F Lok, and H C Thomas. 1985. “Hepatitis B Virus DNA in Saliva , Urine , and Seminal Fluid of Carriers of Hepatitis B e Antigen” 290 (June): 1853–55.
6. Samejima, K., & Earnshaw, W. C. (2005). Trashing the genome: the role of nucleases during apoptosis. Nature Reviews Molecular Cell Biology, 6(9), 677–688. doi:10.1038/nrm1715
7. Alves, Danielle, Gomes Zauli, Carla Lisandre, Paula De Menezes, Cristiane Lommez, De Oliveira, Elvis Cristian, Cueva Mateo, Alessandro Clayton, and De Souza Ferreira. 2016. “Genetics and Molecular Microbiology In-House Quantitative Real-Time PCR for the Diagnosis of Hepatitis B Virus and Hepatitis C Virus Infections.” Brazilian Journal of Microbiology 47 (4): 987–92. https://doi.org/10.1016/j.bjm.2016.07.008.
8.Jain, Surbhi, Ying-hsiu Su, Yih-ping Su, Sierra Mccloud, Ruixia Xue, Tai-jung Lee, Shu-chuan Lin, et al. 2018. “Characterization of the Hepatitis B Virus DNA Detected in Urine of Chronic Hepatitis B Patients,” 19–21.
