Week 2 HW: DNA Design
Repository : Chanda-BiologicalFTTI
Expressing : GFP-ssrA and AmilCP
GFP-ssrA
| iGEM Registry -> BBa_K819007 |
|---|
| 5’ - CTTGTGGCAACAATTTCTACAAACACTTGATACCGTATGAGCATACGGTATAATTGCTTCAACAGAACATATTGACTATCATTAAAGAGGAGAAATACTAGATGCGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATT |
| AGATGGTGATGTTAATGGGCACAAATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCAACATACGGAAAACTTACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCATGGCCAACACTTGTCACTACTTTCGGTTATGGTGTTCAAT |
| GCTTTGCGAGATACCCAGATCATATGAAACAGCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAAAGAACTATATTTTTCAAAGATGACGGGAACTACAAGACACGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTGTTA |
| AGATGGAAGCGTTCAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTACCTGTCCACACAATCTGCCCTTTCGAAAGATCCCAACGAAAAGAGAGACCACATGGTCCTTCTTGAGTTT |
| GTAACAGCTGCTGGGATTACACATGGCATGGATGAACTATACAAAGCTGCTAACGACGAAAACTACGCTCTGGCTGCTTACTAGTAG - ‘3 |
AmilCP
| iGEM Registry -> BBa_K3988019 |
|---|
| 5’ - ATGTCAGTGATCGCTAAGCAAATGACATACAAAGTCTATATGTCTGGGACCGTAAATGGCCACTACTTTGAAGTGGAAGGGGATGGCAAGGGGAAGCCTTACGAGGGTGAACAGACCGTAAAACTGACTGTAACAAAAGGGGGCCCTTTACCTT |
| TCGCATGGGACATCCTTTCCCCTCAATGTCAGTACGGCTCCATCCCTTTTACGAAGTACCCGGAGGATATCCCTGATTATGTTAAGCAGTCTTTCCCAGAAGGCTACACCTGGGAACGGATTATGAATTTCGAGGATGGTGCGGTTTGTACCGTCTCAAAC |
| GACTCATCGATCCAAGGTAACTGCTTCATTTATCACGTTAAATTTTCTGGTCTCAATTTTCCACCTAATGGTCCGGTCATGCAGAAAAAAACCCAAGGGTGGGAGCCAAACACGGAGCGGTTGTTCGCACGCGACGGCATGTTACTCGGGAACAATT |
Host Chasis : E.Coli
| Layer | Part | Type | Source | Justification |
|---|---|---|---|---|
| Sensor | C120 | Promoter | Kernel | Provides continuous expression of the engineered thermal transcriptional regulator so it is available to respond when temperature changes. |
| Sensor | BBa_B0034 | RBS | Kernel | Enables efficient translation of the engineered AsLOV2-EL222 HTH fusion in E. coli. |
| Sensor | Engineered AsLOV2-I532A + EL222 HTH Domain | CDS | Designed | Combines the engineered temperature-responsive AsLOV2-I532A module with the EL222 DNA-binding domain to couple temperature-dependent conformational changes to transcriptional regulation. |
| Sensor | Terminator | Terminator | Kernel | |
| Real-Time Output | C120 | Promoter | Kernel | Provides the promoter activated by the EL222 HTH domain to drive the temperature-responsive reporter. |
| Real-Time Output | BBa_B0034 | RBS | Kernel | Enables translation of the fluorescent reporter in E. coli. |
| Real-Time Output | GFP-ssrA | CDS | Kernel | Provides a fluorescent signal while the thermal response is active, with the ssrA tag promoting degradation after the signal is switched off. |
| Real-Time Output | Terminator | Terminator | Kernel | Terminates transcription of the GFP reporter cassette. |
| Memory | C120 | Promoter | Same as output | |
| Memory | BBa_B0034 | RBS | Kernel | Provides a well-characterized ribosome-binding site for efficient translation initiation in E. coli. |
| Memory | amilCP | CDS | Kernel | Uses the same thermal-responsive regulatory input so that cumulative colour production begins under the same temperature conditions as the fluorescent response. |
| Memory | Terminator | Terminator | Kernel | Provides reliable transcription termination, helping define the end of each expression cassette and reduce unwanted read-through transcription. |
| Vector | E.Coli Backbone | Backbone | Existing vector | Provides the replication origin and selectable marker required to maintain the construct in E. coli. |
Sequences before Fusing both to create Chimeric Protein
Engineered AsLOV2-I532A 5’ GEFLATTLERIEKNFVITDPRLPDNPIIFASDSFLQLTEYSREEILGRNCRFLQGPETDRATVRKIRDAIDNQTEVTVQLINYTKSGKKFWNLFHLQPMRDQKGDVQYFIGVQLDGTEHVRDAAEREGVMLAKKTAENIDEAAKEL ‘3 EL222 HTH Domain 5’ ATGTTGGATATGGGACAAGATCGGCCGATCGATGGAAGTGGGGCACCCGGGGCAGACGACACACGCGTTGAGGTGCAACCGCCGGCGCAGTGGGTCCTCGACCTGATCGAGGCCAGCCCGATCGCATCGGTCGTGTCCGATCCGCGTCTCGCCGACAATCCGCTGATCGCCATCAACCAGGCCTTCACCGACCTGACCGGCTATTCCGAAGAAGAGTGCGTCGGCCGCAATTGCCGATTCCTGGCAGGTTCCGGCACCGAGCCGTGGCTGACCGACAAGATCCGCCAAGGCGTGCGCGAGCACAAGCCGGTGCTGGTCGAGATCCTGAACTACAAGAAGGACGGCACGCCGTTCCGCAACGCCGTGCTCGTTGCACCGATCTACGACGACGACGACGAGCTTCTCTATTTCCTCGGCAGCCAGGTCGAAGTCGACGACGACCAGCCCAACATGGGCATGGCGCGCCGCGAACGCGCCGCGGAAATGCTCAAGACGCTGTCGCCGCGCCAGCTCGAGGTTACGACGCTGGTGGCATCGGGCTTGCGCAACAAGGAAGTGGCGGCCCGGCTCGGCCTGTCGGAGAAAACCGTCAAGATGCACCGCGGGCTGGTGATGGAAAAGCTCAACCTGAAGACCAGCGCCGATCTGGTGCGAATTGCCGTCGAAGCCGGAATCTGA ‘3’
How would you build it?
I would initially build the construct using commercially synthesized DNA fragments followed by Gibson assembly. The construct contains several functional modules: the engineered AsLOV2-I532A–EL222 HTH regulatory fusion, the C120/GFP-ssrA reporter cassette, and the C120/amilCP memory cassette. Rather than synthesizing the entire construct as one long sequence, these modules could be ordered as separate DNA fragments and assembled into an appropriate E. coli backbone. This approach makes it easier to modify or replace individual modules during prototyping.
What does synthesis constrain?
DNA synthesis can introduce sequence errors and may be more difficult for long, repetitive, or structurally challenging sequences. My design is not expected to contain unusually repetitive DNA, but the complete multi-cassette construct is more complex than a single gene. I would therefore use sequence-verified synthetic fragments and verify the assembled construct before testing. If a fragment proved difficult to synthesize accurately, it could be divided into smaller fragments and assembled using Gibson assembly, reducing the length of each individual synthesis target.
Prototype vs. scale
For the prototype, I would build and test a small number of sequence-verified constructs, particularly the I532A AsLOV2 variant, to determine whether the proposed temperature-responsive system produces the expected transcriptional and reporter response.
For larger-scale production, the emphasis would shift toward a standardized, sequence-verified construct and a reproducible DNA manufacturing process. Once the design and regulatory architecture were validated, the same construct could be produced using a scalable synthetic-DNA supplier rather than individually assembling each copy. This separates the exploratory prototype stage from the eventual manufacturing process.
References
- Guerois R, Nielsen JE, Serrano L (2002). Predicting changes in the stability of proteins and protein complexes: a study of more complex cases. Journal of Molecular Biology, 320(2), 369–387.
- Schymkowitz J, Borg J, Stricher F, Nys R, Rousseau F, Serrano L (2005). The FoldX web server: an online force field. Nucleic Acids Research, 33(Web Server issue), W382–W388.
- Harper SM, Neil LC, Gardner KH (2003). Structural basis of a phototropin light switch. Science, 301(5639), 1541–1544.
- Halavaty AS, Moffat K (2007). N- and C-terminal flanking regions modulate light-induced signal transduction in the LOV2 domain of the blue light sensor phototropin 1 from Avena sativa. Biochemistry, 46(49), 14001–14009.
- Zayner JP, Antoniou C, Sosnick TR (2012). The amino-terminal helix modulates light-activated conformational changes in AsLOV2. Journal of Molecular Biology, 419(1–2), 61–74.
- Motta-Mena LB et al. (2014). An optogenetic gene expression system with rapid activation and deactivation kinetics. Nature Chemical Biology, 10, 196–202. (EL222 characterisation)
- Strickland D et al. (2008). Rationally improving LOV domain–based photoswitches. Nature Methods, 5, 267–273. (AsLOV2 fusion design principles)