Part:BBa_K5102073:Design

pRAM_ProgRAM-recording-tape2.0

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal SpeI site found at 18
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal SpeI site found at 18
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 2494
  Illegal BamHI site found at 3554
  Illegal XhoI site found at 2563
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal SpeI site found at 18
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal SpeI site found at 18
  Illegal NgoMIV site found at 4899
  Illegal AgeI site found at 3645
  
• 1000
  COMPATIBLE WITH RFC[1000]

Design Notes

The composite part features several key elements within its modular design:

• *Mammalian promoter*: promoter for plasmid expression of ProgRAM in a mammalian cell line. In the current instance, a CMV enhancer ([BBa_K5102067|https://parts.igem.org/Part:BBa_K5102067]) and a CMV promotor ([BBa_K2217006|https://parts.igem.org/Part:BBa_K2217006]). Alternative mammalian promoters driving the expression are possible, for instance, [BBa_J433025|https://parts.igem.org/Part:BBa_J433025], [BBa_J433001|https://parts.igem.org/Part:BBa_J433001], or [BBa_J433002|https://parts.igem.org/Part:BBa_J433002].
• *5'UTR* (601–680): 5' untranslated region. In the current instance, CMV 5'UTR ([BBa_K5102068|https://parts.igem.org/Part:BBa_K5102068]). This region can be substituted with synthetic alternatives, such as ([BBa_K5102079|https://parts.igem.org/Part:BBa_K5102079] and [BBa_K5102080|https://parts.igem.org/Part:BBa_K5102080]), offering customizable designs for modulation of RNA stability and ribosome recruitment as outlined on the [iGEM Munich 2024 model page|https://2024.igem.wiki/munich/model/#synthetic-5-utr-design].
• *T7 promoter* (681–698): phage T7 promoter ([BBa_K3633015|https://parts.igem.org/Part:BBa_K3633015]) for _in vitro_ expression of the ProgRAM.
• *Recording tape* (700–751): recording tape, functioning as a deamination target and translation initiation site. In the current instance, a 3.0 tape design ([BBa_K5102041|https://parts.igem.org/Part:BBa_K5102041]). This is a recording tape based on the REPAIR system using 30 nt long gRNAs. Alternative designs based on a co-optimal length of 50 nt, are also provided ([BBa_K5102106|https://parts.igem.org/Part:BBa_K5102106] and [BBa_K5102107|https://parts.igem.org/Part:BBa_K5102107]). Alternative 30 nt-based design comprises parts [BBa_K5102072|https://parts.igem.org/Part:BBa_K5102072] to [BBa_K5102078|https://parts.igem.org/Part:BBa_K5102078] The deamination site (writing site), also known as the central base triplet (CBT), was chosen for its high deamination efficacy and fidelity to be at the +22 position. A sequence of 'CAU' was chosen for the CBT following the mammalian Kozak consensus sequence, which results in the deamination environment of moderate efficiency. The reading site was chosen for its high potency towards Cas13b binding disruption to be at the +12 position. The current instance of the tape features 3 intact START codons, corresponding to the initial 0th state, with each START codon having a +1 frame shift relative to its predecessor. The tape was constructed from these minimal sequence constraints, as highlighted on the [iGEM Munich 2024 model page|https://2024.igem.wiki/munich/model/#tape-construction-algorithm].
• *Coding region* (753–4237): _in vivo_ reporter system featuring a fluorescent proteins array. Each array element comprises T2A-XFP-P2A-eUnaG-STOP, with a +1 frame shift relative to its predecessor. The T2A ([BBa_K5102012|https://parts.igem.org/Part:BBa_K5102012], [BBa_K5102013|https://parts.igem.org/Part:BBa_K5102013], and [BBa_K5102014|https://parts.igem.org/Part:BBa_K5102014]) site divides a near-native XFP from the upstream sequences, while the P2A ([BBa_K5102015|https://parts.igem.org/Part:BBa_K5102015], [BBa_K5102016|https://parts.igem.org/Part:BBa_K5102016], and [BBa_K5102017|https://parts.igem.org/Part:BBa_K5102017]) site functions as a separator between the XFP and the eUnaG. This arrangement of 2A peptides was chosen to approach proportionality between expressed proteins. eUnaG ([BBa_K5102009|https://parts.igem.org/Part:BBa_K5102009], [BBa_K5102010|https://parts.igem.org/Part:BBa_K5102010], and [BBa_K5102011|https://parts.igem.org/Part:BBa_K5102011]) is used as total protein expression control and is present in all 3 reading frames. The current instance features miRFP670nano3 ([BBa_K5102001|https://parts.igem.org/Part:BBa_K5102001]), mScarlet3 ([BBa_K5102002|https://parts.igem.org/Part:BBa_K5102002]), and mTagBFP2 ([BBa_K5102003|https://parts.igem.org/Part:BBa_K5102003]) as switching fluorescent proteins indicating the current tape state. Alternative proteins may be utilized if they comply with sequence requirements. The sequence of the current array was enabled by removing hidden stop codons and optimizing for various other parameters, including codon adaptation, minimization of repeats, and exclusion of splice and polyadenylation sites, among others, which is explained in detail on the [iGEM Munich 2024 model page|https://2024.igem.wiki/munich/model/#codon-optimization].
• *3'UTR* (4241–4284): 3' untranslated region. In the current instance, human beta-globin 3'UTR ([BBa_K5102053|https://parts.igem.org/Part:BBa_K5102053]).
• *Aptamer* (4295-4317): an RNA aptamer acting as an anchor, enabling tape pulldown or localization. In the current instance, PP7 phage aptamer ([BBa_K5102054|https://parts.igem.org/Part:BBa_K5102054]).
• *T7 terminator* (4334–4381): phage T7 transcriptional terminator ([BBa_K73172|https://parts.igem.org/Part:BBa_K73172]) for _in vitro_ expression of the ProgRAM.
• *WPRE* (4382–4979): woodchuck hepatitis virus posttranscriptional regulatory element ([BBa_K5102069|https://parts.igem.org/Part:BBa_K5102069]) for increasing transgene expression by minimizing readthrough transcription and improving termination.
• *polyA* (4984–5105): a polyadenylation site for mammalian transcription termination and RNA polyadenylation. In the current instance, SV40 polyA ([BBa_K2217005|https://parts.igem.org/Part:BBa_K2217005]). Alternative polyA sites could be used.

Source

The composite was assembled from gblocks and oligos provided by a DNA synthesis provider.

Cloning of our final constructs, *pRAM_ProgRAM-recording-tapes* were proceeded by many cloning steps. First, it included the creation of a minimal vector for SynBio applications, *pRAM* (BBa_K5102000). The construction of pRAM began with a pcDNA3.4-TOPO vector available to us in the lab and the vector was obtained by several Gibson assembly and KLD reactions. In the end, the backbone includes a CMV enhancer, promoter and 5'UTR, T7 promoter and terminator, Woodchuck posttranscriptional regulatory element (WPRE), SV40 polyA element, plasmid ori, as well as AmpR promoter and CDS for selection. In the end, two BsmBI-v2 recognition sites have been introduced to allow for Golden Gate assembly. Due to the high sequence similarity of the P2A-eUnaG-T2A sequences, cloning of the *pRAM_ProgRAM-recording-tapes* vectors were assembled together in a single tube Golden-Gate reaction using BsmBI-v2 enzyme. The reaction included pRAM backbone, ProgRAM recording tape, and three gBlocks: T2A-miRFP670nano3-P2A-eUnaG, T2A-mScarlet3-P2A-eUnaG, T2A-mTagBFP2-P2A-eUnaG. Following _E. coli_ transformation, eight colonies per construct were picked and screened by colony PCR, using a forward primer binding to the plasmid backbone and a reverse primer complementary to the insert. The results were verified via DNA electrophoresis, and colonies with the expected band size were used to inoculate overnight _E. coli_ cultures. The next day, plasmid DNA was miniprepped and verified by Whole Plasmid Sequencing. <img src="" style="background-color: transparent; width: 100px ;display: block; margin: 0 auto;"/>

References

Truong, D.-J. J. et al. Exonuclease-enhanced prime editors. Nat Methods 21, 455–464 (2024).

Darty, K., Denise, A., & Ponty, Y. (2009). VARNA: Interactive drawing and editing of the RNA secondary structure. Bioinformatics, 25(15), 1974–1975. https://doi.org/10.1093/bioinformatics/btp250

Lorenz, R., Bernhart, S. H., Höner zu Siederdissen, C., Tafer, H., Flamm, C., Stadler, P. F., & Hofacker, I. L. (2011). ViennaRNA Package 2.0. Algorithms for Molecular Biology, 6(1), 26. https://doi.org/10.1186/1748-7188-6-26