Abstract
The principal goal of Bovitect was to develop a proof-of-principle diagnostic device that could detect a range of bTB related biomarkers from biological fluids such as blood or sputum. We have sucessfully achieved this, building a system of parts capable of detecting four immuni response biomarkers present in the blood of infected cattle. This system includes; Cas13a, customised spacer sequences, crRNA and target sequences. Therefore we are submitting this parts collection as one of our gold awards. Below is our collection of parts that other teams can use to recreate our project findings, expand on our prject, or customise the spacer sequence to develop diagnostics for other diseases.
Click to go to a specific collection
A brief explanation of how these parts work in our project:
Gold Award Parts Collection
Our system, based off the SHERLOCK Cas13a system, to detect bovine tuberculosis worked. This meant we had a collection of parts that could be used as a set for either this purpose, or edited to detect other RNA sequences. Because of this, we put forward a collection of our Cas13a coding sequence and T7 inducible promoter, a selection of sgRNA that consists of 5 different spacer sequences attached to the crRNA sequence for Cas13a with a T7 promotor for in-vitro transcription and the 5 sequences containing a complementary target region to these spacers which trigger the positive reaction. On the registry pages themselves is the information of how we selected the sequences and ordered them.
We can tell the Cas13a system worked because we got positive results when the Cas13a, sgRNA, target and probe were added together, but also because we had negative controls that caused no reaction. These were:
- The system missing one of the 4 parts showing all parts are necessary.
- The system with the wrong target showing that the system only activates with the correct RNA sequence.
- The system with the spacer FOSB which folded incorrectly, showing the testing for the folding of sequences was necessary.
Figure 1: A graph of the positive fluorescent results for each target, with PLAUR and CXCL8 working the best and FOSB having a value the same as the negative control.
| Part Number | Part Name | Part Origin | Type | Description + How it was used |
|---|---|---|---|---|
| BBa_K5124001 | LwCas13a codon | Leptotrichia wadei | Basic | An RNA guided enzyme from Leptotrichia wadei that is from a class II, type VI CRISPR system that does not use a trans-activating (tracrRNA). Cas13a recognises and cleaves RNA. Used to detect our RNA targets. |
| BBa_K5124012 | Cas13a crRNA | Leptotrichia wadei | Basic | Codes for the CRISPR-RNA (crRNA) repeat sequence found in the class II, type VI CRISPR loci of Leptotrichia wadei. Combined with each RNA spacer sequence then each combination bound to LwCas13a. |
| BBa_K5124024 | Cas13a CXCL8_target for transcription | Bos taurus | Composite | A 150-nucleotide RNA sequence that, once transcribed, contains a 20-nucleotide sequence that is complimentary to the spacer sequence CXCL8. Has a T7 promoter for transcription. |
| BBa_K5124025 | Cas13a FOSB_target for transcription | Bos taurus | Composite | A 150-nucleotide RNA sequence that, once transcribed, contains a 24-nucleotide sequence that is complimentary to the spacer sequence FOSB. Has a T7 promoter for transcription. |
| BBa_K5124026 | Cas13a NR4A1_target for transcription | Bos taurus | Composite | A 150-nucleotide RNA sequence that, once transcribed, contains a 24-nucleotide sequence that is complimentary to the spacer sequence NR4A1. Has a T7 promoter for transcription. |
| BBa_K5124028 | Cas13a PLAUR_target for transcription | Bos taurus | Composite | A 150-nucleotide RNA sequence that, once transcribed, contains a 24-nucleotide sequence that is complimentary to the spacer sequence PLAUR. Has a T7 promoter for transcription. |
| BBa_K5124029 | Cas13a RGS16_target for transcription | Bos taurus | Composite | A 150-nucleotide RNA sequence that, once transcribed, contains a 24-nucleotide sequence that is complimentary to the spacer sequence RGS16. Has a T7 promoter for transcription. |
| BBa_K5124035 | Cas13a CXCL8 sgRNA | Bos taurus | Composite | Combination of Cas13a crRNA and spacer sequence CXCL8. Repeat sequence folds into a single hairpin loop which is recognised and bound by LwCas13a, leaving the 20-nucleotide spacer sequence free to bind to the target RNA. Has a T7 promoter for transcription. |
| BBa_K5124036 | Cas13a FOSB sgRNA | Bos taurus | Composite | Combination of Cas13a crRNA and spacer sequence FOSB. Repeat sequence folds into a single hairpin loop which is recognised and bound by LwCas13a, leaving the 24-nucleotide spacer sequence free to bind to the target RNA. Has a T7 promoter for transcription. |
| BBa_K5124037 | Cas13a NR4A1 sgRNA | Bos taurus | Composite | Combination of Cas13a crRNA and spacer sequence NR4A1. Repeat sequence folds into a single hairpin loop which is recognised and bound by LwCas13a, leaving the 24-nucleotide spacer sequence free to bind to the target RNA. Has a T7 promoter for transcription. |
| BBa_K5124039 | Cas13a PLAUR sgRNA | Bos taurus | Composite | Combination of Cas13a crRNA and spacer sequence PLAUR. Repeat sequence folds into a single hairpin loop which is recognised and bound by LwCas13a, leaving the 24-nucleotide spacer sequence free to bind to the target RNA. Has a T7 promoter for transcription. |
| BBa_K5124040 | Cas13a RGS16 sgRNA | Bos taurus | Composite | Combination of Cas13a crRNA and spacer sequence RGS16. Repeat sequence folds into a single hairpin loop which is recognised and bound by LwCas13a, leaving the 24-nucleotide spacer sequence free to bind to the target RNA. Has a T7 promoter for transcription. |
| BBa_K5124041 | T7 promoter for in-vitro transcription | T7 Bacteriophage | Basic | A commonly used promoter for protein expression that is recognised by T7 RNA polymerase. Has an additional third G on the 3' end of the promoter. |
| BBa_K5124042 | IPTG inducible T7 promoter system | T7 Bacteriophage | Composite | An IPTG inducible promoter system including a T7 bacteriophage promoter in combination with the E. coli lac operator sequence and the strong RBS from T7 bacteriophage gene 10. |
Cas12a Related Parts
These are parts unrelated to the gold collection. These are based off the DETECTR system which was unsuccessful in our project, so we focused on Cas13a.
| Part Number | Part Name | Part Origin | Type | Description + How it was used |
|---|---|---|---|---|
| BBa_K5124000 | LbCas12a codon | Lachnospiraceae bacterium ND2006 | Basic | An RNA guided enzyme from Lachnospiraceae bacterium ND2006 that is from class II, type V CRISPR system that does not use a trans-activating (tracrRNA). Cas12a recognises and cleaves DNA. Used to detect our DNA targets. |
| BBa_K5124002 | Cas12a EthA_target_b_c | Mycobacterium bovis AF2122/97 | Basic | A 122-nucleotide DNA sequence that contains a 20-nucleotide region that is complimentary to the spacer sequences EthA-b and EthA-c. Used to mimic an infected sample. |
| BBa_K5124003 | Cas12a RD4_target_a | Mycobacterium bovis AF2122/97 | Basic | A 120-nucleotide DNA sequence that contains a 20-nucleotide region that is complimentary to the spacer sequence RD4-a. Used to mimic an infected sample. |
| BBa_K5124004 | Cas12a RD4_target_b_c | Mycobacterium bovis AF2122/97 | Basic | A 122-nucleotide DNA sequence that contains a 20-nucleotide region that is complimentary to the spacer sequences RD4-b and RD4-c. Used to mimic an infected sample. |
| BBa_K5124011 | Cas12a crRNA | Lachnospiraceae bacterium ND2006 | Basic | Codes for the CRISPR-RNA (crRNA) repeat sequence found in the class II, type V CRISPR loci of Lachnospiraceae bacterium ND2006. Combined with each DNA spacer sequence then each combination bound to LBCas12a. |
| BBa_K5124013 | Cas12a ethA_b spacer | Mycobacterium bovis AF2122/97 | Basic | The spacer sequence EthA-b which, once transcribed, is a 20-nucleotide RNA sequence that is complementary to the target bTB DNA. Once attached to the Cas enzyme, binds to the target DNA. |
| BBa_K5124014 | Cas12a ethA_c spacer | Mycobacterium bovis AF2122/97 | Basic | The spacer sequence EthA-c which, once transcribed, is a 20-nucleotide RNA sequence that is complementary to the target bTB DNA. Once attached to the Cas enzyme, binds to the target DNA. |
| BBa_K5124015 | Cas12a RD4_a spacer | Mycobacterium bovis AF2122/97 | Basic | The spacer sequence RD4-a which, once transcribed, is a 20-nucleotide RNA sequence that is complementary to the target bTB DNA. Once attached to the Cas enzyme, binds to the target DNA. |
| BBa_K5124016 | Cas12a RD4_b spacer | Mycobacterium bovis AF2122/97 | Basic | The spacer sequence RD4-b which, once transcribed, is a 20-nucleotide RNA sequence that is complementary to the target bTB DNA. Once attached to the Cas enzyme, binds to the target DNA. |
| BBa_K5124017 | Cas12a RD4_c spacer | Mycobacterium bovis AF2122/97 | Basic | The spacer sequence RD4-c which, once transcribed, is a 20-nucleotide RNA sequence that is complementary to the target bTB DNA. Once attached to the Cas enzyme, binds to the target DNA. |
| BBa_K5124030 | Cas12a ethA_b sgRNA | Mycobacterium bovis AF2122/97 | Composite | Combination of Cas12a crRNA and spacer sequence EthA_b. Repeat sequence folds into a single hairpin loop which is recognised and bound by LbCas12a, leaving the 20-nucleotide spacer sequence free to bind to the target DNA. Has a T7 promoter for transcription. |
| BBa_K5124031 | Cas12a ethA_c sgRNA | Mycobacterium bovis AF2122/97 | Composite | Combination of Cas12a crRNA and spacer sequence EthA_c. Repeat sequence folds into a single hairpin loop which is recognised and bound by LbCas12a, leaving the 20-nucleotide spacer sequence free to bind to the target DNA. Has a T7 promoter for transcription. |
| BBa_K5124032 | Cas12a RD4_a sgRNA | Mycobacterium bovis AF2122/97 | Composite | Combination of Cas12a crRNA and spacer sequence RD4_a. Repeat sequence folds into a single hairpin loop which is recognised and bound by LbCas12a, leaving the 20-nucleotide spacer sequence free to bind to the target DNA. Has a T7 promoter for transcription. |
| BBa_K5124033 | Cas12a RD4_b sgRNA | Mycobacterium bovis AF2122/97 | Composite | Combination of Cas12a crRNA and spacer sequence RD4_b. Repeat sequence folds into a single hairpin loop which is recognised and bound by LbCas12a, leaving the 20-nucleotide spacer sequence free to bind to the target DNA. Has a T7 promoter for transcription. |
| BBa_K5124034 | Cas12a RD4_c sgRNA | Mycobacterium bovis AF2122/97 | Composite | Combination of Cas12a crRNA and spacer sequence RD4_c. Repeat sequence folds into a single hairpin loop which is recognised and bound by LbCas12a, leaving the 20-nucleotide spacer sequence free to bind to the target DNA. Has a T7 promoter for transcription. |
Cas13a Related Parts
These parts are not included in the gold collection but are the basic parts to make up the composite parts of our functional Cas13a system.
| Part Number | Part Name | Part Origin | Type | Description + How it was used |
|---|---|---|---|---|
| BBa_K5124005 | Cas13a CXCL8_target only | Bos taurus | Basic | A 150-nucleotide RNA sequence that, once transcribed, contains a 20-nucleotide sequence that is complimentary to the spacer sequence CXCL8. Used to mimic an infected sample. |
| BBa_K5124006 | Cas13a FOSB_target only | Bos taurus | Basic | A 150-nucleotide RNA sequence that, once transcribed, contains a 24-nucleotide sequence that is complimentary to the spacer sequence FOSB. Used to mimic an infected sample. |
| BBa_K5124007 | Cas13a NR4A1_target only | Bos taurus | Basic | A 150-nucleotide RNA sequence that, once transcribed, contains a 24-nucleotide sequence that is complimentary to the spacer sequence NR4A1. Used to mimic an infected sample. |
| BBa_K5124009 | Cas13a PLAUR_target only | Bos taurus | Basic | A 150-nucleotide RNA sequence that, once transcribed, contains a 24-nucleotide sequence that is complimentary to the spacer sequence PLAUR. Used to mimic an infected sample. |
| BBa_K5124010 | Cas13a RGS16_target only | Bos taurus | Basic | A 150-nucleotide RNA sequence that, once transcribed, contains a 24-nucleotide sequence that is complimentary to the spacer sequence RGS16. Used to mimic an infected sample. |
| BBa_K5124018 | Cas13a CXCL8 spacer | Bos taurus | Basic | The spacer sequence CXCL8 which, once transcribed, is a 20-nucleotide RNA sequence that is complementary to the target bovine mRNA. Once attached to the Cas enzyme, binds to the target RNA. |
| BBa_K5124019 | Cas13a FOSB spacer | Bos taurus | Basic | The spacer sequence FOSB which, once transcribed, is a 24-nucleotide RNA sequence that is complementary to the target bovine mRNA. Once attached to the Cas enzyme, binds to the target RNA. |
| BBa_K5124020 | Cas13a NR4A1 spacer | Bos taurus | Basic | The spacer sequence NR4A1 which, once transcribed, is a 24-nucleotide RNA sequence that is complementary to the target bovine mRNA. Once attached to the Cas enzyme, binds to the target RNA. |
| BBa_K5124022 | Cas13a PLAUR spacer | Bos taurus | Basic | The spacer sequence PLAUR which, once transcribed, is a 24-nucleotide RNA sequence that is complementary to the target bovine mRNA. Once attached to the Cas enzyme, binds to the target RNA. |
| BBa_K5124023 | Cas13a RGS16 spacer | Bos taurus | Composite | The spacer sequence RGS16 which, once transcribed, is a 24-nucleotide RNA sequence that is complementary to the target bovine mRNA. Once attached to the Cas enzyme, binds to the target RNA. |
