Team:CMUQ/Composite Part
Composite Parts
Our composite parts were made by combining the basic parts listed above. By pairing the gRNA fixed loop repeat with segments of the human genome that uniquely represent a wildtype CFTR gene or a mutant variant, we were able to generate guide RNAs that would bind a patient’s DNA if and only if their CFTR gene was of the wildtype variety or a mutated variety.
The collection as a whole paves the way for rapid diagnosis of recessive alleles for cystic fibrosis in patients, whereas the improved part improves our existing methods for diagnosing the recessive sickle cell anemia allele.
gRNA for Cystic Fibrosis (rs75389940_G) (BBa_K2961004)
This guide RNA contains the fixed loop that allows it to combine with a Cpf1 protein molecule. It is then able to use its extended segment (BBa_K2961000) that uniquely binds to the rs75389940 SNP variant of the CFTR gene to draw Cpf1 to that portion of the genome and activate it.
gRNA for wildtype CFTR gene (BBa_K2961005)
Similar to above, this guide RNA contains the fixed loop that allows it to combine with a Cpf1 protein molecule. It is then able to use its extended segment (BBa_K2961001) which uniquely binds to the wild type CFTR gene to draw Cpf1 to this segment of the genome and activate it.
Note that in both cases, it was required that the segment of the gRNA that targets Cpf1 to the DNA was targeting to a segment that was right after the bases “TTTC” (PAM site for Cpf1), which our basic part design took care of.
When Cpf1 is drawn to the appropriate segment of the DNA by its hybridized guide RNA, it is activated. It then cleaves the double stranded DNA at that specific part of the genome where it was targeted, but also indiscriminately cuts any single stranded nucleotide sequence. Thus, we were able to design single stranded fluorescent probes as downstream effectors that offer visual signalling when wildtype or mutant alleles are detected within the human genome.
The combination of tests with the above two composite parts of a human patient DNA sample allows us to quantify the relative amounts of the wild type and mutated alleles that might be present in a patient’s genome. This in turn allows us to predict the patient’s genotype, in terms of the wildtype allele, the rs75389940_G allele, and even any other allele that might be present (labelled abstractly as “other mutant alleles”).
iGEM CMU-Q BioBrick List
| Part Number | Basic Or Composite | Description | Type | Designer | Lengths |
|---|---|---|---|---|---|
| BBa_K2961000 | Basic | CFTR Mutant Gene Segment (rs75389940_G) | DNA | iGEM CMUQ | 24bps |
| BBa_K2961001 | Basic | CFTR Wildtype gene segment | DNA | iGEM CMUQ | 24bps |
| BBa_K2961003 | Basic | gRNA / crRNA fixed-loop repeat for Cpf1 / Cas12a | RNA | iGEM CMUQ | 21bps |
| BBa_K2961004 | Composite | gRNA for Cystic Fibrosis SNP targeting of Cas12a / Cpf1 (rs75389940_G) | RNA | iGEM CMUQ | 45bps |
| BBa_K2961005 | Composite | gRNA for Cystic Fibrosis Wildtype gene targeting of Cas12a / Cpf1 | RNA | iGEM CMUQ | 45bps |
| BBa_K2961006 | Basic | Template DNA sequence encoding for HBB rs334 mutation (A to T) in humans (improves on --> BBa_K2872881) | DNA | iGEM CMUQ | 210bps |