Team:NYU Abu Dhabi/Parts

Parts

Name	Type	Description	Length
BBA_K3289011	DNA	The gene pcaA on locus Rv0470c functions as a cyclopropane mycolic acid synthase. It encodes an S-adenosyl methionine–dependent methyltransferase that catalyzes proximal cyclopropanation of α mycolate, the major mycolic acid subclass of the Mtb cell envelope.	455
BBA_K3289012	DNA	The gene HBcAg codes for a Hepatitis B viral protein which serves as an indicator of active viral replication; meaning that an infected patient with Hepatitis B can likely transmit the virus onto another. HBcAg protein is an antigen that can be found on the surface of the nucleocapsid core (the innermost layer of the Hepatitis B virus).	537
BBA_K3289013	DNA	IS481 is a repetitive, insertable element (transposable element) specific to the Bordetella pertussis pathogen and is used for PCR diagnosis of the disease. The gene can also be found in other members of the genus Bordetella, which cause whooping cough like symptoms.	509
BBA_K3289014	DNA	The gene ypo2088 is specific to Yersinia pestis It is a protein coding gene that makes the enzyme methyltransferase. The enzyme moves methyl groups from one compound to another.	585
BBa_K3289015	Primer	This is the forward PCR primer for the pcAa Mycobacterium tuberculosis gene.	20
BBa_K3289016	Primer	This is the reverse PCR primer for pcAa Mycobacterium tuberculosis gene.	16
BBa_K3289017	Primer	This is the RPA forward primer for pcAa Mycobacterium tuberculosis gene.	32
BBa_K3289018	Primer	This is the reverse RPA primer for pcAa Mycobacterium tuberculosis gene.	32
BBa_K3289019	crRNA	This part is the LbCas12a crRNA for the pcAa Mycobacterium tuberculosis gene.	41
BBa_K3289020	Primer	This is the forward PCR primer for HbcAg Hepatitis B virus gene.	22
BBa_K3289021	Primer	This is the reverse PCR primer for HbcAg Hepatitis B virus gene.	22
BBa_K3289022	Primer	This part is the RPA forward primer for HbcAg Hepatitis B virus gene.	32
BBa_K3289023	Primer	This is the reverse RPA primer for HbcAg Hepatitis B virus gene.	32
BBa_K3289024	crRNA	This part is the LbCas12a crispr RNA for HbcAg Hepatitis B virus gene.	41
BBa_K3289025	Primer	This is the forward PCR primer for the IS481 Bordetella pertussis gene.	22
BBa_K3289026	Primer	This is the reverse PCR primer for the IS481 Bordetella pertussis gene.	22
BBa_K3289027	Primer	This is the RPA forward primer for IS481 Bordetella pertussis gene.	32
BBa_K3289028	Primer	This is the RPA reverse primer for IS481 Bordetella pertussis gene.	32
BBa_K3289029	crRNA	This part is the LbCas12a crispr RNA for IS481 Bordetella pertussis gene.	41
BBa_K3289030	Primer	This is the forward PCR primer for the ypo2088 Yersinia pestis gene.	22
BBa_K3289031	Primer	This is the reverse PCR primer for the ypo2088 Yersinia pestis gene.	22
BBa_K3289032	Primer	This is the RPA forward primer for ypo2088 Yersinia pestis gene.	32
BBa_K3289033	Primer	This is the RPA reverse primer for ypo2088 Yersinia pestis gene.	32
BBa_K3289034	crRNA	This part is the LbCas12a crispr RNA for ypo2088 Yersinia pestis gene.	41

General criteria:

- It must be bacterial or caused by a DNA virus

- It must be a common diseases (at least in one region)

- It must be airborne (also contained in saliva) and contagious

Tuberculosis

one of the top 10 causes of global death (1.6million people died from TB in 2017, 230,000 children)
Global incidence falling at 2% annually, needs to accelerate to 4-5% decline to reach 2020 milestone.
54 million lives saved through TB diagnosis and treatment 2000-2017
Ending TB epidemic by 2030 among health targets of Sustainable Development Goals
Current diagnostic tool Xpert MTB/RIF essay generally takes 2 hours, hard to diagnose drug-resistant strains

Hepatitis B

Viral infection that attacks the liver, leads to chronic infection and high risk of cirrhosis and liver cancer

257 million live with chronic Hep B as of 2015

Virus could survive outside of the body for more than 7 days and cause infection during this time

Diagnosis is usually made through blood tests and our aim is to develop a more accessible diagnosis method

Whooping Cough

Respiratory tract disease caused by pertussis bacteria most dangerous in infants
Spreads easily from person to person by coughing and sneezing
Around 144,000 reported cases in 2017, 89,000 estimated deaths in 2008
Insufficient evidence for vaccine effectiveness in infants

Plague

Caused by bacteria found in fleas and small mammals, symptoms develop after 1-7 day incubation
Infects animals and humans alike, transmitted by flea bites, "direct contact with infected tissues, and inhalation of respiratory droplets"
High case-fatality ratio ("30%-60% for bubonic type, always fatal for pneumonic type when left untreated")
3,248 cases globally 2010-2015, 584 deaths
"Early diagnosis and early treatment can save lives" since antibiotic treatment is effective

References: https://www.who.int/news-room/fact-sheets/detail/tuberculosis https://www.who.int/news-room/fact-sheets/detail/hepatitis-b https://www.who.int/immunization/diseases/pertussis/en/ https://www.who.int/news-room/fact-sheets/detail/plague

PCR Primer Design Process:	gRNA Design Process:	RPA Primer Design Process
NCBI provides Primer-BLAST for automatically designing primers based on a query sequence. To start designing primers, go to the BLAST homepage and scroll down to the Primer-BLAST option under Specialized BLAST. Enter your target sequence either by cut-and-paste or, if it’s listed in NCBI’s databases, as an accession number. The target sequence is normally found earlier on by searching the particular gene sequence and downloading the fasta file or copying directly the sequence. The ‘Descriptions’ section of the report now includes a download menu that provides direct access to FASTA data, either of the complete subject sequence or only the aligned portion, as well as to other formats including GenBank flat files, hit tables and XML. Checkboxes to the left of each subject sequence allow users to download an arbitrary subset of the matching sequences. Clicking the title of a subject now links to the alignment display, which also contains similar download functions for that alignment along with controls to navigate between alignments and back to the ‘Descriptions’ table. In the "search for primers dialogue box", gene sequences from the fasta file was pasted and blasted against all genomes. The length of the primers sequence was unspecified neither was the genome of the organism specified. The primers were designed according to the preset and optimum conditions for Tm. The selection of the right primers for our experiments were done accordingly to the following parameters : GC content = 50% , self complementarity < 3 and complementarity < 8 Preferred primers had the G-C clamp The complementarity test were performed using tools from Oligocalc tools.	For designing the gRNAs, we used benchling. For each DNA sequence we first created our primers (PCR, LAMP, AND RPA) and attached them to our DNA strands in order to be able to visualize them on the software's linear map. Making sure that the RPA and LAMP primers fall within the range of our PCR primers, we then selected as our target an appropriate region that falls within our RPA/LAMP primers in order to create our guide RNAs. When multiple gRNA options were provided, the ones with the highest off-target score were selected. The selected gRNA were then NCBI blasted to make sure that they are specific to the chosen bacterial strain. Some of the gRNAs might as well be specific to other bacterial or viral strains, but since we are testing for only 5 diseases, we made sure that a gRNA that is meant to be specific to one of our strains is not specific to any of the remaining four.	Genes were located in .fasta format on NCBI and sequences were created on Benchling. For RPA primers generated from Primer BLAST, the following values were used in settings according to TwistDx recommendations. Product Size: 100-200, Primer Melting Temperature: 50-100, Primer Size Range: 30-36, Primer Size Optimal: 32, Primer GC Content: 30%-70%. In addition, a Python package called primedRPA (detailed in the paper titled PrimedRPA: primer design for recombinase polymerase amplification assays, available at https://www.ncbi.nlm.nih.gov/pubmed/30101342) was used. The following parameters were used: Desired primer length: 32 Desired probe length: 10 Desired amplicon length: 100 Minimum GC content for primers and probes: 30 Maximum GC content for primers and probes: 70 Tolerated length of the region which could form secondary structure: 5 Tolerated number of background binding nucleotides in primer or probe: 24 Multiple primers were generated and then annotated in Benchling and optimal primers (preferably 1 pair from primer BLAST and 1 from primedRPA) were selected based on location, GC content, fitness within PCR amplicon of the same gene, and quality of available gRNAs within the amplified region of the gene itself as well.

PCR Primer Design Process:

gRNA Design Process:

RPA Primer Design Process

NCBI provides Primer-BLAST for automatically designing primers based on a query sequence.

To start designing primers, go to the BLAST homepage and scroll down to the Primer-BLAST option under Specialized BLAST.

Enter your target sequence either by cut-and-paste or, if it’s listed in NCBI’s databases, as an accession number.
The target sequence is normally found earlier on by searching the particular gene sequence and downloading the fasta file or copying directly the sequence.

The ‘Descriptions’ section of the report now includes a download menu that provides direct access to FASTA data, either of the complete subject sequence or only the aligned portion, as well as to other formats including GenBank flat files, hit tables and XML. Checkboxes to the left of each subject sequence allow users to download an arbitrary subset of the matching sequences. Clicking the title of a subject now links to the alignment display, which also contains similar download functions for that alignment along with controls to navigate between alignments and back to the ‘Descriptions’ table.

In the "search for primers dialogue box", gene sequences from the fasta file was pasted and blasted against all genomes. The length of the primers sequence was unspecified neither was the genome of the organism specified.

The primers were designed according to the preset and optimum conditions for Tm.

The selection of the right primers for our experiments were done accordingly to the following parameters : GC content = 50% , self complementarity < 3 and complementarity < 8

Preferred primers had the G-C clamp

The complementarity test were performed using tools from Oligocalc tools.

For designing the gRNAs, we used benchling. For each DNA sequence we first created our primers (PCR, LAMP, AND RPA) and attached them to our DNA strands in order to be able to visualize them on the software's linear map. Making sure that the RPA and LAMP primers fall within the range of our PCR primers, we then selected as our target an appropriate region that falls within our RPA/LAMP primers in order to create our guide RNAs. When multiple gRNA options were provided, the ones with the highest off-target score were selected. The selected gRNA were then NCBI blasted to make sure that they are specific to the chosen bacterial strain.

Some of the gRNAs might as well be specific to other bacterial or viral strains, but since we are testing for only 5 diseases, we made sure that a gRNA that is meant to be specific to one of our strains is not specific to any of the remaining four.

Genes were located in .fasta format on NCBI and sequences were created on Benchling. For RPA primers generated from Primer BLAST, the following values were used in settings according to TwistDx recommendations. Product Size: 100-200, Primer Melting Temperature: 50-100, Primer Size Range: 30-36, Primer Size Optimal: 32, Primer GC Content: 30%-70%. In addition, a Python package called primedRPA (detailed in the paper titled PrimedRPA: primer design for recombinase polymerase amplification assays, available at https://www.ncbi.nlm.nih.gov/pubmed/30101342) was used. The following parameters were used: Desired primer length: 32 Desired probe length: 10 Desired amplicon length: 100 Minimum GC content for primers and probes: 30 Maximum GC content for primers and probes: 70 Tolerated length of the region which could form secondary structure: 5 Tolerated number of background binding nucleotides in primer or probe: 24

Multiple primers were generated and then annotated in Benchling and optimal primers (preferably 1 pair from primer BLAST and 1 from primedRPA) were selected based on location, GC content, fitness within PCR amplicon of the same gene, and quality of available gRNAs within the amplified region of the gene itself as well.