Team:Florida/Description
Project Inspiration and Description
The SCRIBE system was first created by Fahim Farzadfard and Timothy K. Lu at the Massachusetts Institute of Technology. The inspiration for creating a system like SCRIBE was to be able to do long term recording of a cell’s environment. This biosensora device which uses a living organism or biological molecules to detect the presence of chemicals is useful because it not only is able to detect the presence of extracellular chemicals, but it is able to quantify it as well.
More specifically, the SCRIBE system is induced by chemicals in the environment to make permanent changes in the bacterial chromosomethe main circular genetic part of the bacterial. Normally, to check for these mutations the DNA would have to be sent out for sequencing. However, Team Florida set out to make a system that could check for these mutations in-lab with the use of CRISPR/Cas9CR4 and the PKDsgRNA. By eliminating the necessity to send the DNA out of lab for sequencing, it will economically and practically benefit labs who would like to work with SCRIBE in the future.
The SCRIBE + Cas9 project is exciting for Team Florida because it is a very applicable, mistake-friendly, and diverse project. We say this because it gives amateur microbiologists the ability to couple a chosen stimulus with a desired mutation and quantify that signal. The thought of being able to easily change out different inducible promoterspromoters that can be regulated in the presence of certain factors in SCRIBE, made the idea of using it attractive to Team Florida. We saw applications for its use in discovering concentrations of pollutants in previous iGEM team wikis. However, Team Florida also saw a potential for using the system to quantify the concentrations of heavy metals in supposedly potable drinking water. Our project combines SCRIBE and Cas9, wherein this new tool we will be able to mutate the bacterial chromosome in response to the environmental pollutant and evaluate the concentration of the toxin. We take some inspiration from Team Ionis Paris -2016 who were able to effectively design a bacterial biosensor contained in a drone. Our ultimate goal is to be able to utilize our tool to quantify the concentration of heavy metals in tap water in the state of Florida (and potentially worldwide) that otherwise may go improperly assessed.
We started our project by making the composite parts of our system which consists of SCRIBE , Cas9, and sgRNA. We did not incorporate any heavy metal detecting elements to the system as of yet. SCRIBE consists of the following: Inducible promoter, msr, msd(rpoB), RBS, Reverse Transcriptase, RBS, Constitutive Promoter, Beta Recombinase Protein, and Terminator.
The Promoter, when induced by IPTG begins transcription of the SCRIBE system. Following translation, the reverse transcriptase turns the msr/msd region to from single stranded mRNA to single stranded DNA (ssDNA), with the target rpoB mutation. The Beta Recombinase binds to the ssDNA strand, containing the mutation. Then, it properly inserts it into the Okazaki fragmentshort sequences of DNA nucleotides which are synthesized during lagging strand synthesis into the bacterial chromosome using site specific homologous recombination during chromosomal replication.
Williams, Sarah. "DNA Tape Recorder Stores A Cell's Memories". Science | AAAS, 2019, https://www.sciencemag.org/news/2014/11/dna-tape-recorder-stores-cells-memories.
