Difference between revisions of "Team:Lambert GA/ProjectOverview"

PROJECT OVERVIEW

The identification of pathogens in potentially contaminated sources is crucial for the prevention and treatment of affected individuals from across the world. People in developing nations are particularly distressed by infectious diseases as a consequence of poor sanitation and lack of personal hygiene and access to sufficient resources. Approximately 844 million of these people suffer from a lack of access to safe water, of which 3.4 million succumb to the wide range of infectious diseases transmitted through contaminated water sources [1][2]. Numerous organizations have strived to increase access to clean water in impoverished communities within developing nations. However, pathogens are still able to thrive in water sources, allowing for the emergence of endemics and epidemics that devastate large segments of the population.

One disease in particular, Cholera, is notorious for claiming approximately a million lives annually. While its presence is relatively non-existent in developed nations due to sufficient treatments that are easily accessible, this pathogen devastates communities in developing nations. Current strategies to detect Cholera are severely inadequate and inefficient. Consequently, Cholera cases are prevalent in these communities. Therefore, Lambert iGEM hopes to develop a practical, yet efficient solution to ensure that these epidemics can not only be detected but prevented as well.

Current Methods

With the increasing occurrence of epidemics of Cholera, numerous tools have been developed to detect this pathogen in fecal and water samples. However, they significantly vary in cost and precision, making them incompetent for deployment in the field. The most prevalent detection mechanism for Cholera is the Crystal VC Dipstick, an inexpensive tool that can be easily transported and utilized. However, the accuracy range for this device varies between 60% and 99%, which requires additional lab testing for confirmation and act as a deterrent for guaranteed positive/negative results[4]. Immunoassays are also incorporated for confirmation of the Cholera pathogen. However, the precision of these devices comes at a high cost, as the materials necessary to conduct these tests are expensive and difficult to deploy and transport in a field setting, making it inefficient for testing outside the confines of a laboratory[11]. Another common method involves Polymerase Chain Reaction (PCR), which allows for amplification of the target genes of the Cholera pathogen for confirmation of the O1 and O139 strains. However, incorporation of the PCR method requires the utilization of numerous reagents in the field, in addition to a thermocycler and gel electrophoresis apparatus, making it difficult to exploit in the field[11]. The inefficiency of these methods in terms of costs and/or accuracy has driven the 2018 Lambert iGEM team to develop a novel system capable of delivering accurate and precise results for Cholera identification at a significantly lower cost.

Our Project

This overview explains how the multiple parts of our project work together to be a proactive approach to preventing V. cholerae epidemics using Yemen as a test case. Our process begins with the CALM software predicting outbreaks up to 8 weeks in advance. Our kit with the necessary hardware, software and biosensor cells are pre-deployed to aid workers. Text messages are sent to the aid agencies who notify local workers to deploy the testing kits. Water samples are taken and filtered to extract cells in the size range of V. Cholerae. The cells are lysed and RNA or DNA is extracted (depending whether NASBA is available in field) The RNA/DNA is electroporated into the biosensor cells using our Electropen. Biosensor cells are incubated for 24 hours after which the cell solution is pelleted using the 3-D fuge. The sample is loaded onto the Chrome Q base and using the Color Q App, the results are quantified and uploaded to AWS server which publishes the results. The results from the water sampling feeds into our CALM model completing a feedback loop to ensure continual model improvement.

Recognizing the limitations of the current methods utilized for the detection of Cholera, Lambert iGEM has developed a novel platform to detect and analyze data from this pathogen at a fraction of the cost. While many caveats persist towards pathogen identification in large water sources, access to efficient and inexpensive technology has become the primary prohibitive factor. Inexpensive tests currently utilized for detection have a significant variance in the positive/negative outputs, whereas the precision of other technologies correlates with heightened costs with the lack of access to necessary materials in the field. Lambert iGEM demonstrates the potential for a gene-based detection mechanism for Cholera pathogens that can easily be deployed in the field at drastically lower costs, without sacrificing quality or performance. In tandem, we propose a machine learning model that can predict outbreak inception and spread, allowing for a preventative approach, rather than a reactive one.

Future Implications

While our current technology functions for identification of Cholera, Lambert iGEM hopes to expand this technology to numerous pathogens, establishing a collection of genetic tools for detection and compiling them into a portable synthetic biology toolkit that can be distributed to aid organizations for confirmation of clean water provisions. This detection platform with visible readouts can be integrated into a data collection platform on a global scale, allowing for a proactive response to disease outbreaks and ensuring the safety of the people residing in potentially at-risk areas. Lambert iGEM hopes to revolutionize pathogen detection in order to enhance feasibility, accessibility, affordability, and efficiency.

Cholera Background

Cholera is an acute diarrheal illness caused by an infection of the intestines with the toxigenic bacterium Vibrio cholerae serogroup O1 or O139. V. cholerae O139, first identified in Bangladesh in 1992, has caused numerous outbreaks in the past but recently has only been identified in sporadic cases across Asia. The main form of transmission of the Cholera bacterium is the contamination of water and food by feces from an infected individual. Cholera is prevalent in locations with inadequate water treatment and sanitation infrastructure. The main form of diagnosis for Cholera is a stool sample or rectal swab, which must be sent to a laboratory in order to identify the Cholera bacterium. The key symptom of Cholera is severe diarrhea, which leads to dehydration, pain in the abdominal regions, and lethargy. Approximately one in ten (10%) infected persons will have dire cases of Cholera characterized by watery diarrhea, profuse vomiting, and leg cramps. In these cases, body fluid loss and water-electrolyte imbalance lead to dehydration and shock. Very often, lack of access to treatment can lead to death in a matter of hours. The CDC reports that there are an estimated 2.9 million Cholera cases worldwide.

Unfortunately, even with the existence of Oral Cholera Vaccines (OCVs), an effective tool to combat cholera in developing nations with an 80.2% effectiveness rate, 100,000 Cholera deaths still occur yearly. Other treatments include rehydration therapy, antibiotics, and IV fluids. In the years 2000-2016, the World Health Organization discovered numerous major Cholera epidemics, including Haiti in the Americas, DRC, Somalia and the United Republic of Tanzania in Africa, and Yemen in Asia. These same locations are reported to have poor water infrastructure and lack of access to Cholera treatment centers. As of April 27, 2017, there have been 1,055,788 suspected cases, 612,703 confirmed cases, and 2, 255 deaths from Cholera-related problems. Ultimately, resource allocation has been difficult because Cholera is rapid and sporadic, hindering aid organizations from providing timely solutions.

Timeline

How a Toehold Switch Works

Conventional riboregulators serve many synthetic biology purposes including gene regulation, genetic circuits, and biosensors. However, their low dynamic range and susceptibility to crosstalk inhibit their ability to be sensitive towards RNA recognition. In addition, the significant amount of crosstalk that occurs in natural riboswitches diminishes specificity, which is an important aspect to consider in global applications including diagnostics and biotechnology. To address these limitations, toehold switches have revolutionized engineered and systematic riboregulators through their ability to regulate many cell components at the same time. They exhibit far more favorable reaction kinetics and thermodynamics in addition to their dynamic range and versatility [5][1]. As these synthetic riboregulators utilize linear-linear interactions between RNA, they present a reliable and diverse application in synthetic biology offering a potential role in diagnostics. Thus, the 2018 Lambert iGEM team envisions the use of a toehold switch to create a Cholera detector as one of the propelling motivations for this year’s project.

Toehold switches are riboregulators that activate translation in response to a distinct RNA sequence. It is comprised of a switch and a trigger. The switch is composed of a hairpin loop structure that represses translation through its complementary bases in between the ribosome binding site and start codon, which is followed by a 21 nucleotide linker sequence. These sequences ensure that the toehold switch structure will be maintained while coding for low-molecular weight amino acids that would not interfere with the switch’s function. The toehold domains at the beginning of the hairpin are 12 to 18 nucleotides long and are designed to be complementary to the trigger in order to initiate linear RNA binding [5]. The trigger contains complementary sequences to the toehold domain that once it is in the presence of the switch, it will bind to the hairpin stem and unbind the loop. This exposes the ribosome binding site and start codon, allowing translation of the reporter protein to occur.

The 2018 Lambert iGEM team utilized Toehold Switches in two aspects: characterizing a LacZ toehold switch and developing a Cholera switch.

Cholera Switch

To address the lack of a proactive, feasible detection method for Cholera, Lambert iGEM developed a Cholera toehold switch. One of the largest sources of contracting Cholera is through the consumption of contaminated water. To develop a detection method for these contaminated water sources, Lambert iGEM has located the gene ctxB in the toxigenic bacterium Vibrio cholerae serogroup O1 [3]. This gene encodes for the Cholera enterotoxin subunit B which does not contain toxicity by itself but is specific to the pathogenic strain of Cholera [4]. The use of this gene allows for our designed switch to be an accurate detection mechanism while also addressing any safety issues regarding any contact with toxins.

Using a toehold designer software from the Chinese University of Hong Kong iGEM team, we inputted the RNA sequences of ctxB as the target and other parameters including trigger length, toehold domain length, maximum toehold domain base pairs, working temperature, promoter sequence, and linker sequence. The software then generated possible sequence combinations for each of the switch and triggers while also providing additional data that further characterized each component’s efficacy.

Here is all of our Toehold Construct designs.

We also utilized Nupack: an open source software that analyzes nucleic acid structure in response to a given sequence. By inputting the minimum free energy structures for each of the switch and trigger combinations, Nupack showed the possible formed structure allowing us to determine whether or not each switch and trigger would have a high probability of working. The minimum free energy shown below demonstrates the strength of repression for the switch RNA and the single-strandedness of the trigger RNA for the activated complex [2]. According to Green et al. 2014, a negative ∆GRBS-linker values is correlated to lower switch dynamic range.

Through these two softwares, Lambert iGEM was able to design and test three Cholera switches. These switches all consist of a T7 promoter (Part I719005) along with LacZ (Part I732005) as the reporter gene. We performed a series of experiments to clone our toehold switches.

In order to utilize LacZ color expression as a biosensor mechanism, Lambert iGEM obtained a LacZ toehold construct assembled with a T7 promoter from the Styczynski Lab at the Georgia Institute of Technology. When assembled with a distinct RNA sequence that is complementary to the trigger sequence, the LacZ operon is induced and subsequently breaks down Xgal and produces galactose and a blue pigment. This color can then be characterized by the shade of the blue pigment expression. This part is transformed in pSB3C5 instead of pSB1C3 because when LacZ is induced in a high copy plasmid such as pSB1C3, it drains the metabolism of a cell.







As seen in the figure above, it was observed that the toehold expressed a blue pigment when inoculated into Xgal and Luria Broth (image on the left). Although a lighter shade than when fully induced (image on the right), we hypothesize that this apparent pigment is due to toehold leakiness as a result of the strength of the T7 promoter. The Toehold sequence used in this construct was obtained from the 144 first generation orthogonal toehold switches collection from the 2017 Collins paper titled “ Toehold Switches: De-Novo-Designed Regulators of Gene Expression”. Following this unique toehold sequence is the LacZ operon. We introduced a base pair wobble in the LacZ gene that substituted an Adenine for a Guanine. The wobble mutation sequence was obtained from the Styczynski Lab at the Georgia Institute of Technology and was used to eliminate the illegal EcoRI site in the LacZ operon.

Lambert iGEM utilized a toehold switch and a trigger - which corresponds with RNA sequences of the ctxB1 cholera gene - in a biosensor system. If the trigger sequence is present and binds to the toehold switch, then a blue pigment produced by LacZ appears in the cells.

Water Collection

Prepping the Bottle
1.1 Obtain 4 nalgene bottles (22.75 cm in length, 5.5 cm in width).
1.2 Drill a 1.9 cm (preferably 2 cm) hole in the bottom of the bottle with a ¾ in drill bit.
1.3 Run the bottle through tap water to get any plastic bits out of the bottle. Spray the interior of the bottle with isopropyl alcohol. Rinse with soap and water to remove any alcohol.
1.4 Allow the bottles to dry by placing them on a drying rack.

Prepping Muslin Cloth

1. Obtain 90 cm by 90 cm muslin cloth.
2. Cut the muslin cloth into 6 cm. by 6 cm pieces.
3. Rinse with soap under flowing tap water.
4. Allow the muslin cloth to dry.
5. Fold in half.
6. Place the muslin cloth into an ice bottle.
7. Fill the bottle ¾th of the way with the cloth

Prepping Cheese Cloth

1. Obtain a 5.5 meter by 19.4 cm.
2. Cut the cheese cloth into 20.5 cm by 15 cm.
3. Rinse the cheesecloth in running tap water and soap.
4. Create 3-4 folds and pack the ice bottle.
5. Use a long rod to help push the cloth into the bottle and make the bottle compact.
6. Fill the bottle ¾th of the way with the cloth.

Prepping T-shirt Cloth

1. Obtain 1 t-shirt
2. Cut into 5 cm by 11 cm strips
3. Fold strips 2 times and pack ice bottle
4. Fill bottle ½ way with the cloth

Prepping Coffee Cloth

1. Obtain a pack of large coffee filter
2. Microwave filters to sterilize
3. Cut filters in ½
4. Fold pieces 1-2 times and pack ice bottle
5. Fill bottle ¾th of the way with the filters

Prepping Carb Cloth

1. Add 500 ml of dH20 and 17.5g of agar to a stock jar
2. Autoclave solution
3. Add 500µl of carb
4. Pour plates as usual

Creating Liquid Culture

1. Add 5ml of LB to a 15ml culture tube
2. Inoculate a few colonies of cells

Obtaining Water

1. Obtain 1000 ml of creek water from any nearby water body by placing a stock jar within the water and allowing the water to flow into the stock jar.

Filtration

1. Run distilled water through all the filters until all filters are wet.
2. Get five 600 ml beakers
3. Combine the 5 ml liquid culture of E.coli to the 1000ml creek water. Stir the stock jar until the liquid culture is thoroughly mixed with the creek water.
4. Measure 100ml of the creek water spiked with E. Coli.
5. Run the 100 ml of creek water through the muslin filter bottle. Make sure a labeled 600 ml beaker is placed under the bottle to catch filtered water.
6. Repeat steps 4 and 5 with the negative control (no filter), cheesecloth, t-shirt, coffee filters.
7. Run the creek water spiked with E. coli through the Micron Filter (Positive Control)
1. Pour 100 ml of the creek water spike with E. Coli to the top cup of the micron filter.
2. Attach the filtration vacuum to the side of the micron filter. Turn on the vacuum so the water is filtered through the .2 micron filter.
3. Allow the water to be filtered through the micron filter and stop the vacuum when all the water has filtered to the bottom container of the micron filter.

Serial Dilution and Plating

1. Obtain 6 centrifuge tubes.
2. Plate 150 microliters of each of the filtered solutions in the 6 600ml beakers into corresponding carb plates. Use glass beads to spread the cultures throughout the carb plates.
3. Fill a beaker with 10 mL of distilled water.
4. Pipette 15 microliters of the filtered water that ran through the muslin and dispense in a centrifuge tube. Label the centrifuge tube, muslin.
5. Pipette 185 microliters of distilled water and dispense in the same centrifuge tube.
6. Repeat steps 12 and 13 with the negative control (no filter), cheesecloth, t-shirt, coffee filters, and the micron (positive) filter.
7. Plate 150 microliters of the solutions from the microcentrifuge to the corresponding carb plates. Use glass beads to spread the cultures throughout the carb plates.
8. Place the plates in the incubator and incubate at 37 degrees Celsius for 24 hours.
9. After 24 hours observe the results and note use colony forming units.

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