SCRIBE is a system that mutates the target sequence. When we induceGene activation by a molecule SCRIBE with IPTG, the Lac operon is turned on, providing the host cell with the selected resistanceAntibiotic resistance occurs when bacteria develop the ability to defeat the drugs designed to kill them . The system is then coupled with CRISPR/cas9 which acts as a selection tool to create lethal double stranded breaks in the wild type cells. Therefore, only the mutant cells will remain.

Why is SCRIBE Essential for our Project to Work?

The overall goal of our project is to create a universal selection marker using the SCRIBE and CRISPR/cas9 system. In this way, we can manipulate the SCRIBE system to incorporate any target sequence so that cas9 can efficiently select for mutants that would otherwise be undetected by typical selection markers such as antibiotic resistance or color change.

Components of pFF745 plasmid/SCRIBE (our original plasmid)

-Chloramphenicol resistance gene
-pLac target: encodes target sequence
-Reverse transcriptase: converts the ssRNA into ssDNA.
-β-subunit: binds to the ends of target sequence to prevent degradation of ssDNA

SCRIBE( Synthetic Cellular Recorders Integrating Biological Events) is a tool that utilizes modified retrons that have been transformed into bacterial cells to produce single-stranded DNA in response to a certain stimulus. The ssDNA is incorporated into the genome using the replication system of the bacteria. This technique mutates the bacteria to have rifampicin resistance.The system is then coupled with the enzyme CAS9, that cuts all of the cells that do not contain the mutation. The UF iGEM team for 2019 plans on utilizing SCRIBE and CRISPR/Cas9 to efficiently make and test for mutations. Our first goal was to test the efficacy of the SCRIBE system as demonstrated by team UIUC Illinois (iGEM 2015).

We are replacing the target sequence with rpoB. rpoB mutates the target sequence to include rifampicin resistance. The target DNA is then permanently incorporated it into the E. coli genome.

What’s Happening??

The basic sequence of the SCRIBE system is as follows:

dsDNA → ssRNA → ssDNA

-IPTG IPTG mimics allolactose and triggers transcription of the lac operon acts as the lactose mimic→ lac repressor falls off → RNA polymerase transcribes the rpoB target → turns double stranded DNA into mRNA (single-stranded)
-Reverse transcriptase an enzyme that catalyzes the formation of DNA from an RNA template in reverse transcription converts ssRNA into ssDNA
-Beta recombinase protein catalyzes site-specific recombination binds to the ends of ssDNA to help incorporate the DNA into the bacterial chromosome -Now our cell has our mutation (will now have rifampicin resistance)!

How We Adjusted The SCRIBE System

1.Amplified increase in frequency of a gene or chromosomal region everything on plasmid except for the target sequence
2.Used Circular Polymerase Extension Cloning method used to assemble and clone multiple inserts into a vector in a single step(CPEC) to incorporate rpoB as the target sequence
3.Carried out PCR amplification is the selective amplification of DNA or RNA targets using the polymerase chain reaction to make the plasmid double stranded
4.Transformed genetic alteration of a cell resulting from the direct uptake and incorporation of genetic material from its surroundings plasmids with new insert into dH5-alpha cells
5.Allow E. coli cells time to recover and then plate cells with corresponding antibiotics.
6.Use colony PCR to verify which colonies contained the new insert
-One primer short fragments of single stranded DNA that are complementary to DNA sequences that flank the target region of interest binds to the target sequence and the other primer binds to the backbone.
-Because the forward primer corresponds with the target sequence, plasmids with the insert will express the protein. The mutated plasmid will show bands on the agarose gel.
-If the forward primer does not match up with the target sequence, the protein will not be expressed.
-Plasmids verified with insert were taken for sequencing.
7.Tested optimal time of efficiency for the SCRIBE system when induced with IPTG

Later in the project, we will be incorporating the pFF745 plasmid with CRISPR/Cas 9. Like the SCRIBE plasmid, the cas 9 plasmid also has chloramphenicol resistance. To differentiate between the two components, we altered the SCRIBE plasmid to have kanamycin resistance instead.

Improving the SCRIBE system: Characterization of the Van Promoter

Originally, the J23101 promoter controlled both the reverse transcriptase and Beta subunit sequences. Sometimes, the RNA polymerase will not reach the Beta subunit, leading to the early termination of transcription. To prevent this, we incorporated the Van promoter to be in front of the B-subunit sequence, improving the overall efficiency of the SCRIBE system.

Using CRISPR/Cas9 as a Selection Marker

The goal of this section was to incorporate the guide RNA and Cas 9 into the E. Coli cells already containing the SCRIBE system. The PkdtrpoB565 (guide RNA) binds to the ends of the wild type sequence. The pcas9CR4 is led by the guideRNA to make lethal double stranded breakswhen both strands of the DNA duplex are severed in the wild type sequences. Therefore, the CRISPR/cas9 acts as an efficient selection marker for mutants created by the SCRIBE system.

Team:Florida/Design