Team:Leiden/Parts
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Rapidemic
Parts
Biological engineering differs from other engineering fields like electrical engineering by its lack of functional and structural modularity. When you transfer a gene of one organism to another, its behavior will probably change! iGEM strives to simplify biological engineering through its Registry of Standard Biological Parts, a collection of genetic parts that can be combined to build biological systems. By creating modularity and standardization in this collection of parts, the parts will be more predictable in different contexts. By contributing to this Registry, we hope to share our knowledge and allow anyone to use our parts!
Introduction
One of the parts in the iGEM Registry that is relevant for our project is a G-quadruplex (GQ) DNAzyme, a single-stranded DNA sequence that folds into a G-quadruplex structure and acquires peroxidase activity. The GQ DNAzyme in the Registry was filed by iGEM team Heidelberg 2015 (BBa_K1614007). This sequence is the most widely used GQ DNAzyme1. However, this DNAzyme did not show any peroxidase activity in our experimental conditions. Therefore, inspired by previous findings by Li et al. (2016), we improved this part by adding an adenine residue to the 3' end of its ssDNA sequence to increase its activity and tolerance to lower pH (BBa_K3343001)1. On this page, we describe in detail how we improved this part.
We also characterized a new part (BBa_K3343000). This is the GQ DNAzyme with highest peroxidase-mimicking activity reported in literature (2016)1. Firstly, we compared this DNAzyme with DNAzymes BBa_K1614007 and BBa_K3343001 in phosphate buffer pH 6 to test which would be most effective in our diagnostic kit. DNAzyme BBa_K3343000 indeed was the most potent DNAzyme (Fig. 1). Secondly, we confirmed its peroxidase-mimicking activity in a range of buffers of different pH (Results page Fig. 8b). Lastly, the kinetic parameters (molar maximum TMB conversion rate (kcat) and TMB affinity (Km)) of the part were obtained by varying the substrate concentration in the pH 6 phosphate buffer (Results page Fig. 7 and Table 1). These parameters were implemented in our (semi-)empirical model to simulate the reaction kinetics of our technology. For the characterization of this new part, please visit the Results. page or the iGEM Registry page.
Improving an existing part
Unfortunately, the GQ DNAzyme in the Registry(BBa_K1614007), filed by iGEM team Heidelberg 2015, did not show any peroxidase activity in our experimental conditions. Presumably, this is because we performed the oxidation reaction at pH 6, at which the activity of the DNAzyme is very low1. The optimal pH of the part is approximately 8.5, and its activity sharply decreases to a very low rate (0 to 1 µM/min) when the pH decreases from 8 to 61,2.
In our project, a low pH was necessary to decrease the reducing power of DTT in the oxidation reaction mix. DTT is a reducing agent, which inhibits the accumulation of oxidized product in the reaction catalyzed by the DNAzyme. We could not prevent DTT's presence in the oxidation reaction mix, and thus we had to decrease the pH to reduce its activity.
Thus, we attempted to improve the original part by increasing its tolerance to lower pH conditions necessary for our reactions. Inspired by literature, we did this by adding an extra adenine to the 3' end of the ssDNA sequence1.
Results
We compared the peroxidase-mimicking activity of the original part (BBa_K1614007) and our improved part (BBa_K3343001) at pH 6. n 0.1 M phosphate buffer at pH 6. The experimental procedures are described on the Experiments page. A third DNAzyme (BBa_K3343000) was included as extra control; this is the DNAzyme with the highest activity reported in literature (2016)1. This DNAzyme has an intrinsic 3' adenine, but differs from the improved part (BBa_K3343001) in its backbone sequence. The sequences of the three DNAzymes can be found in Table 1. A negative control was included with pH 6 phosphate buffer in place of DNAzyme.
Table 1. GQ DNAzyme ssDNA sequences
| DNAzyme | Description | Sequence (3' to 5') |
|---|---|---|
| BBa_K1614007 | Original part, most widely used DNAzyme | GGGTAGGGCGGGTTGGG |
| BBa_K3343001 | Improved part with additional adenine | GGGTAGGGCGGGTTGGGA |
| BBa_K3343000 | DNAzyme with highest activity reported in literature | CTGGGAGGGAGGGAGGGA |
The peroxidase-mimicking activity of three GQ DNAzymes was obtained by measuring the absorbance at 650 nm over time (Fig. 1). The activity of the original part (BBa_K1764007) was similar to the blank, indicating no or very low peroxidase-mimicking activity at pH 6.0. The activity of the improved part (BBa_K3343001) was much higher, comparable to DNAzyme BBa_K3343000.
Fig. 1 Peroxidase-mimicking activity of three DNAzymes registered as BBa_K1614007 (original part), BBa_K3343001 (improved part with additional adenine) and BBa_K3343000 (DNAzyme with highest activity reported in literature).
Conclusion
By adding one adenine residue to its 3' end, we greatly improved the peroxidase-mimicking activity of the existing part BBa_K1614007 in phosphate buffer pH 6. Further research into a wider range of buffers and different pH would greatly contribute to the characterization of this improved part. Nevertheless, these results helped our project by extending the number of GQ DNAzyme sequences that could be used for the proof-of-concept of our technology. Moreover, by providing a part with a broader pH range, it offers more possibilities and applications for future teams.
Amplification primers and targets
Table 2 and 3 contain the synthetic parts that were used as target sequences and primers for our RPA reactions.
Table 2. Target sequences for different organisms.
| Target | Target name | Sequence (5' to 3') |
|---|---|---|
| Saccharomyces cerevisiae | RPA_temp_SC | AGCATGAGAGCTTTTACTGGGCAAGAAGACAAGAGATGGAgaataCAGCCGGGCCTGCGCTTAAGTGCGCGGTCTTGCTAGGCTTGTAAGTTTCTTTCTTGCTATTCCAAACGGTGAGAGATTTCTGTGCTTTTGTTATAGGACAATTAAAACCGTTTCAATACAACACACTGTGGAGTTTTCATATCTTTGCAACTTTTTCTTTGGGCATTCGAGCAATCGGGGCCCAGAGGTAACAAACACAAACAATTTTATCTATTCATTAAATTTTTGTCAAAAACAAGAATTTTCGTAACTGGAAATTTTAAAAATATTAAAAACTTTCAACAACacaatTCTTGGTTCTCGCATCGATGAAGAACGCAGCGAAATGCGATACGTAATGTGAATTGCAGAATTCCGTGAATCATCGAATCTTTGAACGCACATTGCCCCCTTGGTATTCCAGGGGGCATGCCTGTTTGAGCGTCATTTCCTTCTCAAACATTCTGTTTGGTAGTGAGTGATACTCTTTGGAGTTAACTTGAAATTGCTGGCCTTTTCATTGGATGTTTTTTTTCCAAAGAGAGGTTTCTCTGCGTGCTTGAGGTATAATGCAAGTACGGTCGTTTTAGGTTTTACCAACTGCGGCTAATCTTTTTTATACTGAGCGTATTGGAACGTTATCGATAAGAAGAGAGCGTCTAGGCAACAATGTTCTTAAAGTCCCTATAGTGAGTCGTATTAGCGC |
| Bacillus subtilis | RPA_temp_BS | TTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAATCCTAGAGATAGGACGTCCCCTTCGGGGGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACAGAACAAAGGGCAGCGAAACCGCGAGGTTAAGCCAATCCCACAAATCTGTTCTCAGTTCacttcGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGTAATCGCGGtatAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTTAGGAGCCAGCCCCTATAGTGAGTCGTATTAGCGC |
| Plasmodium falciparum | RPA_temp_PF | CACGGGAAAACTCACTAGTTTAAGACAAGAGTAGGATTGACAGATTAATAGCTCTTTCTTGATTTCTTGGATGGTGATGCATGGCCGTTTTTAGTTCGTGAATATGATTTGTCTGGTTAATTCCGATAACGAACGAGATCTTAACCTGCTAATTAGCGGTAAGTACACTATATTTTTATTTGAAATTGAATATAGGTAATTATACATGTTTATTCAGTGTTCAAATTAGGATATTTTTTTATTAAAATATTCTTTTCCCTGTTCTACTATAATAATTTGTTTTTTTTTACTCTATTTCTCTCTTCTTTTAAGAATGTACTTGTTTGATTAAATAAAGCTTCTTAGAGGAACAGTGTGTATCTAACACAAGGAAGTTTAAGGCAACAACAGGTCTGTGATGTCCTTAGATAAACTAGGCTGCACGCGTGCTACAATGATATATATAACAAGTTGTTAAAAATGTACTTATAAATAAGTGTGTACAGTTTTTCCTGTACTGAAAAGTATAGGTAATCTTTATCAGTATATATCGTAATTGGGATAGATTATTGCAATTATTAATCTTGAACGAGGAATGCCTAGTAAGCATGATTCATTAGATTGTGCTGACTACGTCCCTGCCCTTTGTACCCCTATAGTGAGTCGTATTAGCGC |
| Mycobacterium bovis | RPA_temp_MB | CCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCGACGCCGCGTGGGGGATGACGGCCTTCGGGTTGTAAACCTCTTTCACCATCGACGAAGGTCCGGGTTCTCTCGGATTGACGGTAGGTGGAGAAGAAGCACCGGCCAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCGAGCGTTGTCCGGAATTACTGGGCGTAAAGAGCTCGTAGGTGGTTTGTCGCGTTGTTCGTGAAATCTCACGGCTTAACTGTGAGCGTGCGGGCGATACGGGCAGACTAGAGTACTGCAGGGCAGACTGGAATTCCTGGTGTAGCGGTGGAATGCGCAGATATCAGGAGGAACACCGGTGGCGAAGGCGGGTCTCTGGGCAGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGGTGGGTACTAGGTGTGGGTTTCCTTCCTTGGGATCCGTGCCGTAGCTAACGCATTAAGTACCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGGAATTGACGGGGGCCCGCACAACCCTATAGTGAGTCGTATTAGCGC |
| Influenza A H1N1 | RPA_temp_IA | GTTCAACACTTCCCAGAAGATCTGGTGCCGCAGGTGCTGCGGTGAAAGGAGTTGGAACAATAGCAATGGAGTTAATCAGAATGATCAAACGTGGAATCAATGACCGAAATTTCTGGAGGGGTGAAAATGGACGAAGGACAAGGGTTGCTTATGAAAGAATGTGCAATATCCTCAAAGGAAAATTTCAAACAGCTGCCCAGAGGGCAATGATGGATCAAGTAAGAGAAAGTCGAAACCCAGGAAACGCTGAGATTGAAGACCTCATCTTCCTGGCACGGTCAGCACTTATCCTGAGGGCATCAGTTGCACATAAATCATGTCTGCCTGCTTGTGTATATGGGCTTGCAGTAGCAAGTGGCCATGACTTTGAAAGGGAAGGGTACTCACTGGTCGGGATAGACCCATTCAAATTACTCCAAAACAGCCAAGTGGTCAGCCTGATAAGACCAAATGAAAACCCAGCTCACAAGAGTCAATTGGTGTGGATGGCATGCCACTCTGCTGCATTTGAGGATTTAAGAGTGTCAAGTTTCATAAGAGGAAAGAAAGTGATTCCAAGAGGAAAGCTTTCCACAAGAGGGGTCCAGATTGCTTCAAATGAGAATCCCTATAGTGAGTCGTATTAGCGC |
Table 3. Primer sequences for different targets.
| Target | Primer set | Primer | Primer name | Sequence (5' to 3') |
|---|---|---|---|---|
| Saccharomyces cerevisiae (BY4741) | SC1 | Forward | SC1_F_p1 | ACATTCTGTTTGGTAGTGAGTGATACTCTT |
| Reverse | SC1_R | TCTTATCGATAACGTTCCAATACGCTCAGT | ||
| SC1_R_Nt.AlwI_EAD2+3A | TCCCTCCCTCCCTCCCAGAGATGATCCTCTTATCGATAACGTTCCAATACGCTCAGT | |||
| SC1_R_Nt.BsmAI_EAD2+3A | TCCCTCCCTCCCTCCCAGCGAGACTCTTATCGATAACGTTCCAATACGCTCAGT | |||
| SC1_R_Nt.BspQI_EAD2+3A | TCCCTCCCTCCCTCCCAGCGAAGAGCTCTTATCGATAACGTTCCAATACGCTCAGT | |||
| SC1_R_Nt.BstNBI_EAD2+3A | TCCCTCCCTCCCTCCCAGTCCAGACTCTCTTATCGATAACGTTCCAATACGCTCAGT | |||
| SC2 | Forward | SC2_F_p2_exp | GTAATGTGAATTGCAGAATTCCGTGAATCA | |
| Reverse | SC2_R_exp | CAAGCACGCAGAGAAACCTCTCTTTGGAAAA | ||
| SC2_R_exp_Nt.AlwI_EAD2+3A | TCCCTCCCTCCCTCCCAGAGATGATCCCAAGCACGCAGAGAAACCTCTCTTTGGAAAA | |||
| SC2_R_exp_Nt.BsmAI_EAD2+3A | TCCCTCCCTCCCTCCCAGCGAGACCAAGCACGCAGAGAAACCTCTCTTTGGAAAA | |||
| SC2_R_exp_Nt.BspQI_EAD2+3A | TCCCTCCCTCCCTCCCAGCGAAGAGCCAAGCACGCAGAGAAACCTCTCTTTGGAAAA | |||
| SC2_R_exp_Nt.BstNBI_EAD2+3A | TCCCTCCCTCCCTCCCAGTCCAGACTCCAAGCACGCAGAGAAACCTCTCTTTGGAAAA | |||
| SC3 | Forward | SC3_F_p34 | GGGCCTGCGCTTAAGTGCGCGGTCTTGCTA | |
| Reverse | Reverse SC3_R | TGTTTGTTACCTCTGGGCCCCGATTGCTCG | ||
| SC3_R_Nt.AlwI_EAD2+3A | TCCCTCCCTCCCTCCCAGAGATGATCCTGTTTGTTACCTCTGGGCCCCGATTGCTCG | |||
| SC3_R_Nt.BsmAI_EAD2+3A | TCCCTCCCTCCCTCCCAGCGAGACTGTTTGTTACCTCTGGGCCCCGATTGCTCG | |||
| SC3_R_Nt.BspQI_EAD2+3A | TCCCTCCCTCCCTCCCAGCGAAGAGCTGTTTGTTACCTCTGGGCCCCGATTGCTCG | |||
| SC3_R_Nt.BstNBI_EAD2+3A | TCCCTCCCTCCCTCCCAGTCCAGACTCTGTTTGTTACCTCTGGGCCCCGATTGCTCG | |||
| SC4 | Forward | SC4_F_p34 | CTGCGCTTAAGTGCGCGGTCTTGCTAGGCT | |
| Reverse | SC4_R | GTGTTTGTTACCTCTGGGCCCCGATTGCTC | ||
| SC4_R_Nt.AlwI_EAD2+3A | TCCCTCCCTCCCTCCCAGAGATGATCCGTGTTTGTTACCTCTGGGCCCCGATTGCTC | |||
| SC4_R_Nt.BsmAI_EAD2+3A | TCCCTCCCTCCCTCCCAGCGAGACGTGTTTGTTACCTCTGGGCCCCGATTGCTC | |||
| SC4_R_Nt.BspQI_EAD2+3A | TCCCTCCCTCCCTCCCAGCGAAGAGCGTGTTTGTTACCTCTGGGCCCCGATTGCTC | |||
| SC4_R_Nt.BstNBI_EAD2+3A | TCCCTCCCTCCCTCCCAGTCCAGACTCGTGTTTGTTACCTCTGGGCCCCGATTGCTC | |||
| Bacillus subtilis | BS1 | Forward | BS1_F_p1 | CTTGACATCCTCTGACAATCCTAGAGATAG |
| Reverse | BS1_R | TCATAAGGGGCATGATGATTTGACGTCATC | ||
| BS1_R_Nt.AlwI_EAD2+3A | TCCCTCCCTCCCTCCCAGAGATGATCCTCATAAGGGGCATGATGATTTGACGTCATC | |||
| BS1_R_Nt.BsmAI_EAD2+3A | TCCCTCCCTCCCTCCCAGCGAGACTCATAAGGGGCATGATGATTTGACGTCATC | |||
| BS1_R_Nt.BspQI_EAD2+3A | TCCCTCCCTCCCTCCCAGCGAAGAGCTCATAAGGGGCATGATGATTTGACGTCATC | |||
| BS1_R_Nt.BstNBI_EAD2+3A | TCCCTCCCTCCCTCCCAGTCCAGACTCTCATAAGGGGCATGATGATTTGACGTCATC | |||
| BS2 | Forward | BS2_F_p2 | TGACAAACCGGAGGAAGGTGGGGATGACGT | |
| Reverse | BS2_R | CCGCGATTACTAGCGATTCCAGCTTCACGC | ||
| BS2_R_Nt.AlwI_EAD2+3A | TCCCTCCCTCCCTCCCAGAGATGATCCCCGCGATTACTAGCGATTCCAGCTTCACGC | |||
| BS2_R_Nt.BsmAI_EAD2+3A | TCCCTCCCTCCCTCCCAGCGAGACCCGCGATTACTAGCGATTCCAGCTTCACGC | |||
| BS2_R_Nt.BspQI_EAD2+3A | TCCCTCCCTCCCTCCCAGCGAAGAGCCCGCGATTACTAGCGATTCCAGCTTCACGC | |||
| BS2_R_Nt.BstNBI_EAD2+3A | TCCCTCCCTCCCTCCCAGTCCAGACTCCCGCGATTACTAGCGATTCCAGCTTCACGC | |||
| Mycobacterium bovis | MB1 | Forward | MB1_F_p1 | ACGGTAGGTGGAGAAGAAGCACCGGCCAAC |
| Reverse | MB1_R | CGAACAACGCGACAAACCACCTACGAGCTC | ||
| MB1_R_Nt.AlwI_EAD2+3A | TCCCTCCCTCCCTCCCAGAGATGATCCCGAACAACGCGACAAACCACCTACGAGCTC | |||
| MB1_R_Nt.BsmAI_EAD2+3A | TCCCTCCCTCCCTCCCAGCGAGACCGAACAACGCGACAAACCACCTACGAGCTC | |||
| MB1_R_Nt.BspQI_EAD2+3A | TCCCTCCCTCCCTCCCAGCGAAGAGCCGAACAACGCGACAAACCACCTACGAGCTC | |||
| MB1_R_Nt.BstNBI_EAD2+3A | TCCCTCCCTCCCTCCCAGTCCAGACTCCGAACAACGCGACAAACCACCTACGAGCTC | |||
| MB2 | Forward | MB2_F_p2 | CCGCGGTAATACGTAGGGTGCGAGCGTTGT | |
| Reverse | MB2_R | GTTCCTCCTGATATCTGCGCATTCCACCGC | ||
| MB2_R_Nt.AlwI_EAD2+3A | TCCCTCCCTCCCTCCCAGAGATGATCCGTTCCTCCTGATATCTGCGCATTCCACCGC | |||
| MB2_R_Nt.BsmAI_EAD2+3A | TCCCTCCCTCCCTCCCAGCGAGACGTTCCTCCTGATATCTGCGCATTCCACCGC | |||
| MB2_R_Nt.BspQI_EAD2+3A | TCCCTCCCTCCCTCCCAGCGAAGAGCGTTCCTCCTGATATCTGCGCATTCCACCGC | |||
| MB2_R_Nt.BstNBI_EAD2+3A | TCCCTCCCTCCCTCCCAGTCCAGACTCGTTCCTCCTGATATCTGCGCATTCCACCGC | |||
| Plasmodium falciparum | PF1 | Forward | PF1_F_p1 | GCTCTTTCTTGATTTCTTGGATGGTGATGC |
| Reverse | PF1_R | TATAGTGTACTTACCGCTAATTAGCAGGTT | ||
| PF1_R_Nt.AlwI_EAD2+3A | TCCCTCCCTCCCTCCCAGAGATGATCCTATAGTGTACTTACCGCTAATTAGCAGGTT | |||
| PF1_R_Nt.BsmAI_EAD2+3A | TCCCTCCCTCCCTCCCAGCGAGACTATAGTGTACTTACCGCTAATTAGCAGGTT | |||
| PF1_R_Nt.BspQI_EAD2+3A | TCCCTCCCTCCCTCCCAGCGAAGAGCTATAGTGTACTTACCGCTAATTAGCAGGTT | |||
| PF1_R_Nt.BstNBI_EAD2+3A | TCCCTCCCTCCCTCCCAGTCCAGACTCTATAGTGTACTTACCGCTAATTAGCAGGTT | |||
| PF2 | Forward | PF2_F_p2 | CAGGTCTGTGATGTCCTTAGATAAACTAGG | |
| Reverse | PF2_R | AGGCATTCCTCGTTCAAGATTAATAATTGC | ||
| PF2_R_Nt.AlwI_EAD2+3A | TCCCTCCCTCCCTCCCAGAGATGATCCAGGCATTCCTCGTTCAAGATTAATAATTGC | |||
| PF2_R_Nt.BsmAI_EAD2+3A | TCCCTCCCTCCCTCCCAGCGAGACAGGCATTCCTCGTTCAAGATTAATAATTGC | |||
| PF2_R_Nt.BspQI_EAD2+3A | TCCCTCCCTCCCTCCCAGCGAAGAGCAGGCATTCCTCGTTCAAGATTAATAATTGC | |||
| PF2_R_Nt.BstNBI_EAD2+3A | TCCCTCCCTCCCTCCCAGTCCAGACTCAGGCATTCCTCGTTCAAGATTAATAATTGC | |||
| Influenza A H1N1 | IA1 | Forward | IA1_F_p1 | CTTCCTGGCACGGTCAGCACTTATCCTGAG |
| Reverse | IA1_R | GACTCTTGTGAGCTGGGTTTTCATTTGGTC | ||
| IA1_R_NtAlwI_EAD2+3'A | TCCCTCCCTCCCTCCCAGAGATGATCCGACTCTTGTGAGCTGGGTTTTCATTTGGTC | |||
| IA1_R_NtBsmAI_EAD2+3'A | TCCCTCCCTCCCTCCCAGCGAGACGACTCTTGTGAGCTGGGTTTTCATTTGGTC | |||
| IA1_R_NtBspQI_EAD2+3'A | TCCCTCCCTCCCTCCCAGCGAAGAGCGACTCTTGTGAGCTGGGTTTTCATTTGGTC | |||
| IA1_R_NtBstNBI_EAD2+3'A | TCCCTCCCTCCCTCCCAGTCCAGACTCGACTCTTGTGAGCTGGGTTTTCATTTGGTC |
References
- Li, W. et al. (2016). Insight into G-quadruplex-hemin DNAzyme/RNAzyme:adjacent adenine as the intramolecular species forremarkable enhancement of enzymatic activity. *Nucleic Acids Research*: 44(15), p. 7373-7384.
- http://parts.igem.org/Part:BBa_K1614007