Part:BBa_K2785007
Single Cell Recording (4 Repeat)
Our designed system of chronological event recording consists of a CRISPR/Cas9 base editor, four sgRNAs controlled by separate inducible promoters, and a recording plasmid containing repeating units of DNA. gRNA A and gRNA B alternate under the absence and presence of blue light to direct the base editor to make a mutation on the recording plasmid at the current frame. Once one mutation is made, this allows gRNA A or B to come along and recognize the next frame. By constantly shifting the frame that is able to record, the system is keeping track of elapsed time. Under the presence of a stimulus, both gRNA stimulus A and gRNA stimulus B are produced and depending on the current frame of DNA, the gRNA directs the base editor to mark the current frame with a unique mutation. The unique mutation changes the sequence to a restriction enzyme site.
This construct is our recording plasmid which contains the target sequence and 3 repeating units of DNA.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Single Cell Recording (4 Repeat)
Our designed system of chronological event recording consists of a CRISPR/Cas9 base editor, four sgRNAs controlled by separate inducible promoters, and a recording plasmid containing repeating units of DNA. gRNA A and gRNA B alternate under the absence and presence of blue light to direct the base editor to make a mutation on the recording plasmid at the current frame. Once one mutation is made, this allows gRNA A or B to come along and recognize the next frame. By constantly shifting the frame that is able to record, the system is keeping track of elapsed time. Under the presence of a stimulus, both gRNA stimulus A and gRNA stimulus B are produced and depending on the current frame of DNA, the gRNA directs the base editor to mark the current frame with a unique mutation. The unique mutation changes the sequence to a restriction enzyme site.
This construct is our recording plasmid which contains the target sequence and 3 repeating units of DNA.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Team UFLORIDA Contribution:
Due to very limited amount of time given in lab, team UFLORIDA has taken the opportunity to contribute to iGEM by adding current literature of bio-parts.
For this project Team UFlorida believes this recording base excision mechanism can be improved by using a different promoter from the PlacO promoter. The Ptac promoter(BBa_K3910997) is a hybrid promoter combining the -35 trp operon region and -10 region of lacUV5. The promoter is IPTG inducible. Based on the Voigt's 2021 lab paper on plasmid and promoter quantifiable activity, pTac shows to be a strong inducible promoter, reaching its highest transcript copy number at around 100 uMolar concentration of promoter. This system was coupled with a strong B0064 Ribosome Binding Site:BBa_B00 64. A strong ribosome binding site(BBa_K3910998) will allow the machinery of E.coli to bind to a sequence that due to its strong nature, it will have better binding affinity to the ribosome and as such better protein expression as shown in the paper.
We also believe that by adding a riboJ ribozyme (BBa_K3424025) sequence downstream of the Ptet-O right before the CDS of the system sites will also allow for better expression of the Base Editor system as well. Insulators like riboJ work by creating hairpin formations in the untranslated region of a circuit, allowing the desired CDS sequence to be better expressed by reducing gene expression issues that can arise, specially using heterologous genetic circuits.
Sources:
Shao, B., Rammohan, J., Anderson, D. A., Alperovich, N., Ross, D., & Voigt, C. A. (2021, March 5). Single-cell measurement of plasmid copy number and promoter activity. Nature News. Retrieved October 19, 2021, from https://www.nature.com/articles/s41467-021-21734-y#Sec21.
https://parts.igem.org/wiki/index.php?title=Part:BBa_K3424025
A plasmid toolbox for controlled gene expression across ... (n.d.). Retrieved October 19, 2021, from https://www.researchgate.net/publication/352397681_A_plasmid_toolbox_for_controlled_gene_expression_across_the_Proteobacteria.
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