Team:SUNY Oneonta/Poster

Poster: SUNY Oneonta iGEM Team

CONFIRMING A2 ALLELES USING LUMINESCENCE IN THE FIELD

Presented by Team SUNY_Oneonta 2020

Asana Ibrahim1,3, Ashley Germosen Rosa1,4, Britney Cordice-Little1,3, Claire Curtin1,3, Eduardo Lopez1,3, Helen Sanchez1,4, Luke Boylan-Hockett1,5, Madalyn Hammes1,3,4, Sean Walis1,3, Jill Fielhaber2,3 & Kelly Gallagher2,4

1iGEM Team Member, 2Team Advisor, 3Department of Biology, 4Department of Chemistry and Biochemistry, 5Department of Music. State University of New York College at Oneonta, Oneonta, NY, USA.

Abstract:

Small dairy farmers in upstate New York are struggling to survive in a factory-farming marketplace. To increase profits, some farmers are producing specialty products; one such product in the US is A2 milk. A2 milk differs from the more common A1 milk in the sequence of beta-casein, one of the main milk proteins. The gene that codes for the A1/A2 alleles of beta-casein differs by a single nucleotide polymorphism (SNP). A2 milk is growing in popularity in the US due to purported health benefits. Our team aims to create a field-deployable genetic test to facilitate breeding of A2 herds. Our system utilizes a 5’ Flap endonuclease (Flappase) and quenched-oligonucleotides that differentially bind to the A1/A2 alleles of beta-casein. When in the correct conformation, Flappase will cleave the oligonucleotides, dequenching the fluorescent tag. Dairy farmers using this system will be able to rapidly identify cows carrying the A2 allele for breeding.

Project Inspiration

About SUNY Oneonta’s local community:

  • Located in rural Otsego county, Upstate New York (population density 62/square mile (24/km2) (Figure 1).
  • Agriculture is an important industry in Otsego County, where farmland makes up 30% of the total area.
  • From 2007-2017, Otsego County lost 100 farms- this is a problem!
  • We decided to focus on dairy farms since New York State is 4th in milk production, coming to 2.5 billion dollars in 2017 alone.

Figure 1. Map of New York State. Otsego county is highlighted in red. The position of Oneonta is indicated with a cow icon.

A project inspired by farmers:

  • We interviewed two local farmers, John Tauzel and Jeff Darling, and learned about the challenges small dairy farmer face (Figure 2).

Figure 2. Small, family owned and operated farms in our area face many challenges.

  • They also introduced us to some of the approaches to mitigating these problems (Figure 3).

Figure 3. Strategies to improve profit margins.

And we learned about the emerging A2 milk market.

A2 milk:

  • A2 milk is an emerging market in the United States.
  • The difference between A1 milk, which is predominant in the U.S., and A2 milk is a single amino acid in beta-casein (a protein that makes up 30% of all milk proteins).
  • This difference is created by a Single Nucleotide Polymorphism (SNP) in the beta-casein gene (Figure 4).
  • Some studies suggest that there might be health benefits to consuming A2 milk over the A1 form.

Figure 4. The alleles of the beta-casein gene (CSN2). The alleles differ at an SNP in exon 7 of this gene. This SNP creates an amino acid substitution. The presence of a Histidine in the A1 allele product creates a cleavage site, that produces a peptide Beta-caseomorphin 7 (BCM7). Some studies have suggested (controversially) that BCM7 had negative health effects (1,2)

Synthetic biology can help dairy farmers:

  • A2 milk is sold in the US by the a2 Milk Company™. They specialize in organic milk.
  • Only cows that are homozygotes for the A2 allele produce milk that contains only the A2 beta-casein variant.
  • A2 homozygous cows are rare in the US. Most breeds are heterozygotes. Therefore, A2 cows must be bred (Figure 5).
  • For farmers to breed A2 cows, genetic testing is required. This can be slow and expensive, and requires sending samples to a third party.
  • We were inspired to help farmers bypass this.

Figure 5. Breeding A2 cows from heterozygotes results in a 25% chance of an A2 cow.

The goal of our project is to use synthetic biology to create an on-site detection system that can be used to rapidly detect the “A status” of cattle.

References:

  1. Caroli, A. M., Chessa, S., & Erhardt, G. J. (2009). Invited review: milk protein polymorphisms in cattle: effect on animal breeding and human nutrition. Journal of dairy science, 92(11), 5335–5352. https://doi.org/10.3168/jds.2009-2461
  2. Martin, P., Szymanowska, M., Zwierzchowski, L., & Leroux, C. (2002). The impact of genetic polymorphisms on the protein composition of ruminant milks. Reproduction Nutrition Development,42(5), 433-459. doi:10.1051/rnd:2002036 
Project Overview and Goals

PROJECT OVERVIEW AND GOALS

The main goal of our project is to develop a field-deployable genetic testing system to detect the presence or absence of the A2 allele of the β-casein (CSN2 gene). This will allow farmers to genetically identify their cows and facilitate selective breeding for cows with an A2 CSN2 genetic background.

Our method must be able to detect a single nucleotide polymorphism (SNP) that differentiates the A1 and A2 alleles of CSN2. To create the detection system, we decided the following workflow (Figure 1).

Figure 1. Summary of the strategy employed to create a field-genotyping system for the A1 and A2 alleles of the CSN2 gene.

Goal 1: Amplification

To detect the SNPs, we needed an amplification process to create an enriched sample of the CSN2 gene. We elected to use Recombinase Polymerase Amplification. RPA uses recombinase to amplify DNA in a primer-dependent manner. It differs from traditional amplification techniques because of its efficiency and isothermal properties. This will allow the detection system to operate in the field without a thermocycler.

Goal 2: Genotyping

The inspiration for our genotyping system was the Invader Assay. This system uses a structure-specific 5’ flap endonucleases (Flappases) to cleave overlapping oligonucleotides at the target DNA containing an SNP. The flappase assay is another one tube reaction which can be adapted to field testing at room temperature for our farmers. While the use of flap endonucleases has been validated for SNP genotyping, there is no flap endonuclease in the iGEM tool kit, inspiring us to develop BioBrick standard flappase parts.

Goal 3: Implementation

Although we’ve selected molecular mechanisms for genotyping that are field-friendly, we still need to develop devices for temperature control and fluorescent signal detection

that farmers can use to conduct the test. We elected to create a simple heat block and have devised a plan to create a cell phone based fluorometer.

Goal 4:Education

We expended a significant amount of effort to promote synthetic biology in our community through a three-week educational outreach program, a virtual science camp for middle school students. Not only did our work with the camp teach families in our area about synthetic biology, it also provided valuable feedback on the design of our heat block device.

Details of our work on each goal can be found in the other sections of this poster.

RPA Engineering

Sourcing CSN2 DNA: 

  • A positive control DNA for each of the CSN2 alleles is needed.
  • The A1/A2 SNP is in Exon 7.
  • The A1 version contains a A/T base pair, while the A2 version contains a C/G base pair at base 74 in exon 7 .

Figure 1. Structure of the CSN2 gene. Introns are represented as a gray line, exons as a blue box. Exon lengths and the location of the A1/A2 allele SNP are also depicted.

  • The A1 and A2 versions of exon 7 were synthesized and cloned into pSB1C3.

Figure 2. Colony PCR to confirm cloning of the A2 allele of CSN2 exon 7. The A2 allele of exon 7 was synthesized and then cloned into pSB1C3. E. coli DH5α were transformed with the constructs and colony PCR was performed to confirm presence of the insert.

  • The A2 allele insert was successfully cloned into pSB1C3 (Figure 2).
  • We were not able to confirm the presence of the A1 allele (data not shown).

Primer Design and Optimization of RPA:

  • 18 primer pairs were designed. (link to ZIP Archive of Primer Documentation)
  • Primers were checked for hairpins, self and heterodimerization.
  • Three forward primers: 2F, 4F, 18F; and three reverse primers: 6R, 10R, 11R, were selected for testing (Figure 3).

Figure 3: Primers selected for testing in RPA Amplification.

  • Primers were tested by RPA using a 40-minute amplification performed at 37 °C (Figure 4).

Figure 4. RPA primer optimization. Combinations of forward and reverse RPA primers were used to perform RPA on A2 exon 7 positive control DNA. RPA products were subjected to agarose gel electrophoresis.

  • Primer pairs 2F:10R, and 4F:11R were selected for further optimization of the RPA protocol.
  • RPA reaction temperature was altered to assess primer performance (Figure 5).

Figure 5. RPA temperature optimization. RPA reactions using the indicated primer pairs were run at 37°C or 42 °C for 40 minutes. RPA products were subjected to agarose gel electrophoresis.

  • Pair 2F, 10R works best at 37°C and pair 4F, 11R works well at both temperatures.
  • Pair 4F, 11R yielded additional bands of a higher than predicted molecular weight at 37°C.

Future work:

  • Cloning and validation of A1 allele positive control DNA.
  • Isolate and develop field procedures for extracting bovine genomic DNA.
  • Complete optimization of RPA with positive control DNA (primer concentration, reaction time).
  • Develop and test RPA procedures with genomic DNA samples.
Homology Model of Yeast Flappase (RAD27)

Reasons to produce a homology model:

  • The structure of the yeast enzyme is unknown .
  • To provide evidence that methods used with the human enzyme (FEN-1) would be transferable to yeast Flappase.
  • To be able to rationally optimize interactions with target oligonucleotides and performance of the Flappase system in the future(g., designing mutations).

Methods and Resources Used to Produce and Validate the Model:

  • The UniProt database was used to obtain the amino acid sequence of the yeast Flappase, called RAD27 (5).
  • BLAST was used to compare the yeast sequence to proteins of known structure in order to identify possible templates (1). We decided to build two models based on the high sequence identity to the human (Figure 1A) and furiosus (Figure 1B) Flappases.
  • SWISS-MODEL was used to produce the models and analyze their validity (3).
  • UCSF Chimera was used to produce images of our models and analyze the structures via Ramachandran plot (2).

Figure 1. Flappase Sequence Alignments. (A) Sequence alignment of yeast and human Flappase, (B) yeast and P. furiosus Flappase.

Results:

  • Our homology model is shown in Figure 2.
  • Statistical measures of model quality indicate the structure is reasonable (Table 1).
  • As expected, due to the much higher level of sequence identity, the model based on the human structure is of slightly better quality. We will use this model in future work.

Figure 2. Views of the model from two different angles. The yeast flappase homology model (tan) is superimposed on the human FEN-1 structure (light blue).

Table 1

Template Structure

Sequence Identity

Quality Data

Ramachandran Results

P. furiosus

(PDB ID: 1B43)

43.08%

QMEAN: ( -3.12)

GMQE: (0.56)

Favored: 90.14%

Outlier: 2.9%

Homo Sapiens

(PDB ID: 5UM9)

62.31%

QMEAN: (-2.48)

GMQE: (0.61)

Favored: 92.75%

Outlier: 2.42%

- GMQE (ranges from 0 to 1.0, a higher number is a more reliable model)
- QMEAN (the closer to 0, the higher the model quality; numbers < -4 indicate poor model quality)

Figure 3. Ramachandran plots of the yeast flappase model based on human FEN-1 (left) and P. furiosus FEN-1 (right)

Future Work:

  • Further validate the model via simulation.
  • Build a target nucleic acid into the model.
  • Use the model to interpret functional data from our system, once obtained.

References

  1. Camacho, C., Coulouris, G., Avagyan, V., Ma, N., Papadopoulos, J., Bealer, K., & Madden, T. L. (2009). BLAST+: architecture and applications. BMC bioinformatics, 10, 421. https://doi.org/10.1186/1471-2105-10-421
  1. Pettersen, E. F., Goddard, T. D., Huang, C. C., Couch, G. S., Greenblatt, D. M., Meng, E. C., & Ferrin, T. E. (2004). UCSF Chimera--a visualization system for exploratory research and analysis. Journal of computational chemistry, 25(13), 1605–1612. https://doi.org/10.1002/jcc.20084
  1. Waterhouse, A., Bertoni, M., Bienert, S., Studer, G., Tauriello, G., Gumienny, R., Heer, F. T., de Beer, T., Rempfer, C., Bordoli, L., Lepore, R., & Schwede, T. (2018). SWISS-MODEL: homology modelling of protein structures and complexes. Nucleic acids research, 46(W1), W296–W303. https://doi.org/10.1093/nar/gky427
  1. UniProt Consortium (2019). UniProt: a worldwide hub of protein knowledge. Nucleic acids research, 47(D1), D506–D515. https://doi.org/10.1093/nar/gky1049
Flappase and Parts

Design of the Flappase System:

  • Detection system is based on use of a 5’ Flap Endonuclease (Flappase).
  • Two DNA oligonucleotides (a spacer and a flap) are used to target DNA.
    • Spacer: Anneals upstream of the SNP and has a single 3’ nucleotide complementary to the SNP (cSNP).
    • Flap: contains the following three sections:
  1. A 5’ flap portion, that is not complementary to the target sequence.
  2. A single nucleotide, complementary to the cSNP (ccSNP).
  3. A 3’ targeting section, which is complementary to the target gene.
  • The Flappase will specifically recognize the flap and spacer oligos when they form a Holliday junction-like structure and cleave the flap.
  • The 5’ and 3’ ends of our Flap oligo are tagged with a fluorophore-quencher pair. Cleavage of the flap will release the fluorophore from quenching, yielding a signal.

Design of Flap and Spacer Oligonucleotides:

  • We designed two sets, one to target the A1 allele and one for the A2 allele of CSN2 (Figure 1).

Figure 1. A1 and A2 flap and spacer oligo design. One spacer and one flap oligo were designed to target the A1 and A2 alleles of CSN2. Features of the oligos are highlighted to correspond to the different regions (orange and green represent the SNP and SNP binding nucleotides, dark blue represents the oligo sequences that anneal to the template, light blue represents the 5’flap portion)

Flappase Part Design:

  • Because yeast grow well and retain enzyme function at a wide range of temperatures, we used the Flappase gene from Saccharomyces cerevisiae (a.k.a., RAD 27).
    • We constructed the basic part BBa_K3389002, which contains the flappase coding sequence.
    • To create the composite part BBa_K3389003 we added a 10X His tag to the C-terminal domain of the RAD27 gene, a strong RBS from the iGEM toolkit, and a T7 promoter to allow us to adjust expression of the gene (Figure 2).

Figure 2. Expression system for Rad27 Flappase contains a T7 promoter and a strong RBS to regulate expression of Rad27, 10X His tag (for purification,) and double stop codon.

  • Composite part was transformed into coli BL21 DE3 cells.
  • Cloning success was verified by colony PCR and restriction digestion (Figure 3).

Figure 3. Verification of the Flappase expression construct. The Flappase construct was designed and ligated into pSB1C3. Ligations were then transformed into E.coli BL21. (A) Colonies were screened using colony PCR to identify clones containing the insert. (B) Minipreps and restriction digestions were used to confirm insert.

Future Work:

  • Develop protocols for inducing Flappase expression in coli, such as lysing the cells, and purification via metal affinity gravity-flow column purification
  • Begin developing and optimizing the Flappase assay, beginning with a draft protocol based on literature reports of Flappase assays using the human enzyme.
Implementation Plan

Selection of a fluorophore:

  • Researched potential fluorophore-quencher pairs.
  • Compiled information on the quantum yield and absorption/emission spectra of each (Table 1).

Table 1. Potential fluorophore and quencher pairs considered (1).

Quantum yield of fluorophore

Fluorophore

(absorbance max.,
 excitation max. in nm)

Quencher

(absorbance max., range in nm)

0.92

Alexa Fluor. 488 (492,517)

Iowa Black FQ (531,420-520)

?

Rhodamine green-X (504,531)

Iowa Black FQ (531,420-520)

0.9

ATTO 532 (534,554)

Black Hole Quencher 1 (534,480-580)

0.8

ATTO 550 (560,575)

Black Hole Quencher 1 (534,480-580)

0.92

ATTO 565 (570,591)

Black Hole Quencher 2 (579, 560-670)

0.8

ATTO Rho101 (592,609)

Black Hole Quencher 2 (579, 560-670)

0.79

Alex Fluor. 546 (556,571)

Black Hole Quencher 1 (534,480-580)

0.66

Alexa Fluor. 594 (584,616)

Black Hole Quencher 2 (579), 560-670

0.61

Alexa Fluor. 532 (527,553)

Black Hole Quencher 1 (534,480-580)

0.9

6-FAM, fluorescein (495,520)

Black Hole Quencher 1 (534,480-580)

0.1

TAMRA (559,583)

Black Hole Quencher 1 (534,480-580)
  • Black Hole Quencher 1 and 6-FAM were selected due to their compatible quenching range and the high quantum yield of 6-FAM at 0.9.

Planning Construction of a Fluorometer:

  • Determined that the fluorometer must meet the following specifications:
  • Easy to use
  • Affordable
  • Portable
  • Reliable
  • Made from commercially available parts
  • Provide direct access to results
  • A published and validated design of a cellphone-interfaced spectrophotometer serves as inspiration.

Figure 2. Schematic design of a cellphone-based spectrophotometer (2).

  • Construction of the fluorometer will begin in part two of the project. Construction will involve:
  • Constructing the body of the fluorometer using plans adapted from (2).
  • Adding excitation and emission filters to make the system ready for 6-FAM
  • Creating or adapting open-source software to create a control/display app in collaboration with Team Moscow-Russia.

Construction and testing of a heating device:

  • Regulated temperature is necessary for RPA and the Flappase assay.
  • Commercial heat blocks are expensive.
  • A cost-effective heat block system was prototyped based on published designs (3), (Figure 2).

Figure 3. Heat Block Prototype. The prototype is made from common materials, purchased on Amazon (Peltier chip, aquarium thermostat, AC adaptor), or built by us (milled block of aluminum, 3D printed block and chip chamber).

  • The heat block performed well in initial lab testing.
  • The design was tested as part of the Go STEM “Engineering with Biology” Camp.
  • Performance of the heat blocks was variable.
  • Many of the Peltier chips we distributed in the kit were unreliable; the temperature of would fluctuate by several degrees.

 

Future work:

  • Test fluorophore signal intensity in Flappase assay.
  • Prototype, test, and refine cell phone fluorometer.
  • Iterate heat block design Peltier chips from different manufacturer.

 

References:

  1. Integrated DNA Technologies - Fluorophores Modification. (n.d.).  Retrieved October 25, 2020, from www.idtdna.com/site/Catalog/Modifications/Dyes.
  2. Laganovska, K., Zolotarjovs, A., Vázquez, M., Donnell, K. M., Liepins, J., Ben-Yoav, H., . . . Smits, K. (2020). Portable low-cost open-source wireless spectrophotometer for fast and reliable measurements. HardwareX,7. doi:10.1016/j.ohx.2020.e00108 
  3. Maloney, D., Says:, E., Says:, R., Says:, W., & Says:, L. (2018, November 13). Hacked Heating Instruments For The DIY Biology Lab. Retrieved October 26, 2020, from https://hackaday.com/2018/11/12/hacked-heating-instruments-for-the-diy-biology-lab/
Educational Outreach
  • We partnered with CDO-STEM and the A.J. Read Science Discovery Center of SUNY Oneonta to develop and host a 3-week virtual camp for middle school aged students.

Figure 1: Poster advertising Engineering with Biology, a three-week virtual STEM Summer Camp.

Click Here to See The Camp's Schedule

Development of a Go STEM kit:

  • Put together a kit of materials for hands on activities (Figure 2).
  • Examples of materials for individual activities included in the packages included:
    • Constructing a candy model of DNA
    • Building and testing a heat block – this activity was used to test the functionality of the device discussed in Implementation
    • Transforming coli DH5alpha
    • At home agarose gel electrophoresis
  • Team members tested materials provided in the kits to work out protocols and ensure funtionality.

Figure 2. Assembling and testing materials for the Go-STEM kit. (A) Materials for the DIY heat block. (B) Equipment for at home agarose gel electrophoresis. (C) Kit materials for transforming E. coli. (D) Mady, Helen and Chloe test an at home agarose gel electrophoresis activity using sugar free lemonade as the running buffer.

Creation of Educational Materials and Activities:

  • Developed a Bingo game about Synthetic Biology and iGEM.
    • This activity was used to review concepts previously covered in the camp.
  • Created an iGEM Scavenger hunt.
    • This activity was used to introduce participants to the breadth of issues that synthetic biology can address.
    • Campers learned how diverse the iGEM program is and that students from across the world are all completing interesting and innovative projects.
  • We gave a presentation about our project, Ca2LF, to the camp.
    • This tied the activities they participated in at home to the lab, and the use of synthetic biology to improve lives in our area.

See Our Scavenger Hunt Activity
See Our Bingo Activity

Figure 3: One slide from our presentation to the GoSTEM Camp about our project.

Leading Activities and Mentoring Participants:

  • We lead small group activities (Figure 4), such as:
    • An engineering design activity, in which participants applied the engineering design process to create a superhero team, which were then tested in a disaster role playing game.
    • A synthetic biology ethics activityadapted from the Building with Biology  public forum, “Editing Our Evolution” (2).
  • We staffed “help-lines”, through which camp participants could contact us for assistance as they worked on at home, hands-on-activities.

Figure 4: Asana and Chloe lead a superhero simulation to test characters engineered by campers.

References:

  1. Editing Our Evolution: Rewriting the Human Genome forum. (n.d.). Retrieved October 25, 2020, from https://www.nisenet.org/catalog/editing-our-evolution-rewriting-human-genome-forum