Synthesize any piece of DNA you want! DNA synthesizer middleman - PowerPoint PPT Presentation

“Synthesize any piece of DNA you want!”

DNA ¡synthesizer ¡ • middleman • time consuming • toxic reagents • inefficient http://www.gotosam.com/ABI_3400.JPG

• Terminal deoxynucleotidyl Transferase - natively found in mammalian immune cells - codon-optimized and expressed in E. coli - does not require template to add nucleotides • Reversible Heat Labile Protected Nucleotides - 3’proteted group decouples at 95˚C

G T TdT C A A A

CleanAmp ¡dNTP ¡Trial ¡ Normal ¡dNTP ¡

CleanAmp ¡dNTP ¡Trial ¡ Normal ¡dNTP ¡

Dipping ¡Arm ¡ Peristals8c ¡Pumps ¡ Reac8on ¡ Chamber ¡ Heated ¡Water ¡Baths ¡

• Two degrees of freedom • Uses two NEMA-17 motors, lasercut panels, and 3D printed gears • Cycles reaction chamber between water baths

• Two 3D printed parts, 4 bearings, 5 screws • Total Cost: <$18 • Compatible with any NEMA-17 Stepper Motor

“White paper” on our round table discussion on Biosafety, Biosecurity and Bioethical ramifications of the De novo Enzyme Mediated DNA Synthesizer and Safety Strain yeast projects.

• Create a safety strain for cells used in production. • Use telomere lengths to control the lifespans of organisms that can be used in production • Model safety strain system in Saccharomyces cerevisiae

When cells undergo cell division, their telomeres shorten. When the telomeres have reached a certain length, the cell will reach senescence and die.

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EST2 ¡ 5 ¡

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RAD52 ¡ EST2 ¡ 5 ¡

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RAD52 ¡ EST2 ¡ 5 ¡ VPS75 ¡ MAK31 ¡

~45 bp ~45 bp 5’ 3’ (EST1, RAD52, EST2, MAK31, VPS75) 5’ 3’ 3’ Protocol Protocol ~45 bp ~45 bp 5’ primer (TRP1, LEU2, KAN, URA) primer 3’ 5’

● Proceeded to do single knockouts. ● VPS75 knocked out using TRP following a LiAc Yeast Transformation Procedure.

Procedure ● Made growth curves for VPS75, RAD52, and EST2. Confirmed backup recovery. ● Proceeded to make double knockouts. ● EST2: RAD52 Double knockout. ─ Growth curve proved that a double knockout occurred. ● More efficient method: Sporulation

● Low cost test tube roller ● 12 sample capacity ● Easily customizable for various tube sizes ● Uses laser cut panels and a 3D printed hub

● Relatively low-cost open- source centrifuge ($260) ● Max Speed: 9000 RPM ● Utilizes easily sourced parts ● Battery powered, portable

● Knockout of SIR1 Gene ● Galactose Activated Cre Recombinase Switch Cohen, ¡H., ¡and ¡Sinclair, ¡D.A. ¡(2001) ¡Recombina8on-­‑mediated ¡lengthening ¡of ¡terminal ¡repeats ¡requires ¡ the ¡Sgs1 ¡DNA ¡helicase. ¡PNAS ¡

● Build a linked two plasmid system ● Create bio-system whose inputs and outputs can be easily swapped ● Introduce students to synthetic biology ● No need for high-tech, expensive equipment

1. Ligated input and 2. Co-transformed 3. Tested System! output composite in E.Coli parts into Backbone

● GFP was expressed in the plasmid exposed to UV twice as much ● Possibility for a leaky system-try varying strengths of phage activator/promoter system ● Possibly switch to a high copy plasmid for more expression

● The GFP composite part under the PO promoter from the 2007 Cambridge IGEM team (BBa_I746321) was further characterized by our system by acting as the input plasmid for our dual plasmid system. ● When shocked with UV, the UV promoter activated the phage activator tin our biobricked part and turned on the PO phage promoter in the output plasmid that activated the GFP reporter.

Input Promoters Output Reporters

• Can accommodate up to 30 samples simultaneously • Durable interlocking plate construction

Outreach ¡

Thank ¡you ¡