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

A A A C T G

TdT

CleanAmp ¡dNTP ¡Trial ¡ Normal ¡dNTP ¡

CleanAmp ¡dNTP ¡Trial ¡ Normal ¡dNTP ¡

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

• 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
• rganisms 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.

5 ¡

5 ¡

EST2 ¡

5 ¡

5 ¡

EST2 ¡ RAD52 ¡

5 ¡

5 ¡

EST2 ¡ RAD52 ¡ VPS75 ¡ MAK31 ¡

(EST1, RAD52, EST2, MAK31, VPS75)

5’ 3’

(TRP1, LEU2, KAN, URA)

5’ 3’ 3’ 3’ 5’ 5’

~45 bp ~45 bp ~45 bp ~45 bp

Protocol primer Protocol primer

• 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

• ccurred.
• 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

• 2. Co-transformed

in E.Coli

• 3. Tested System!
• 1. Ligated input and
• utput composite

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

• utput plasmid that activated the GFP reporter.

Input Promoters Output Reporters

• Can accommodate up to 30

samples simultaneously

• Durable interlocking plate

construction

Outreach ¡

Thank ¡you ¡