Part:BBa_K5102072:Design

pRAM_ProgRAM-recording-tape1.0

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 and a CMV promotor BBa_K2217006. Alternative mammalian promoters driving the expression are possible, for instance, BBa_J433025, BBa_J433001, or BBa_J433002.
• 5'UTR (601–680): 5' untranslated region. In the current instance, CMV 5'UTR BBa_K5102068. This region can be substituted with synthetic alternatives, such as BBa_K5102065, offering customizable designs for modulation of RNA stability and ribosome recruitment.
• T7 promoter (681–698): phage T7 promoter 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 1.0 tape design BBa_K5102033. 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 and BBa_K5102107. Alternative 30 nt-based design comprises parts BBa_K5102072 to 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, BBa_K5102013, and BBa_K5102014 site divides a near-native XFP from the upstream sequences, while the P2A BBa_K5102015, BBa_K5102016, and 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, BBa_K5102010, and BBa_K5102011 is used as total protein expression control and is present in all 3 reading frames. The current instance features miRFP670nano3 BBa_K5102001, mScarlet3 BBa_K5102002, and mTagBFP2 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.
• 3'UTR (4241–4284): 3' untranslated region. In the current instance, human beta-globin 3'UTR 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.
• T7 terminator (4334–4381): phage T7 transcriptional terminator BBa_K731721 for in vitro expression of the ProgRAM.
• WPRE (4382–4979): woodchuck hepatitis virus posttranscriptional regulatory element 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. Alternative polyA sites could be used.

Structural modelling of tape’s 3’UTR

ProgRAM is a complex system, yet it offers significant modularity and supports the incorporation of various add-ons, as outlined on the Design page. This flexibility is particularly evident in the 3’ UTR, which can accommodate additional elements with minimal impact on deamination or reporter expression. This region of the RNA is highly suitable for introducing RNA elements such as barcodes, ribozymes, or RNA aptamers. To ensure that the ProgRAM design can integrate elements into its 3’UTR while preserving their structural integrity, we conducted an in silico study. In this analysis, we structurally modeled our tape with the addition of the Okra aptamer system (BBa_K5102070), which we obtained as a tool for RNA visualization and quantification. The Okra aptamer is a bright and stable aptamer designed for mRNA imaging. Our structural modeling (as demonstrated with Okra-appended BBa_K5102073) confirmed that all eight fluorophore-binding sites in the 796 bp 4xdOkra region were preserved in their original conformation, with minimal structural abberations.

Visualisation of ViennaRNA folding results (Lorenz et al., 2011) of BBa_K5102072 using VARNA (Darty et al., 2009). RNA tape regions are highlighted as follows (from 5’ to 3’): 5’UTR in gray, recording tape 2.0 in gold, miRFP670nano3 in dark red, mScarlet3 in bright red, mTagBFP2 in light blue, eUnaG in pale green, WPRE in magenta, 4xdOkra in purple.

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.

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