SynBio Is Expensive iGEM is great. Brings synthetic biology to the - - PowerPoint PPT Presentation

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SynBio Is Expensive iGEM is great. Brings synthetic biology to the - - PowerPoint PPT Presentation

Aug 09, 2023 162 likes •442 views

SynBio Is Expensive iGEM is great. Brings synthetic biology to the masses. But its really expensive. Enzymes, Digestions, Ligations Equipment - Thermocycler, Incubator, Centrifuge Minipreps, Gels,etc Want to help other teams as well! We

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SynBio Is Expensive

iGEM is great. Brings synthetic biology to the masses. But it’s really expensive. Enzymes, Digestions, Ligations Equipment - Thermocycler, Incubator, Centrifuge Minipreps, Gels,etc Want to help other teams as well! We decided to look at DIY approaches to synthetic biology to make iGEM accessible.

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DIY Cost Comparison

Protocol # Price Transformations 4 $8.25 Plating 4 $0.71 Overnights 4 $0.15 Digests 4 $0.80 Ligations 1 $6.40 Minipreps 4 $1.74 Gels 2 $2.70

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Safety

Emerging new field of biotechnology - a new hope, a new worry… Our Project:

  • Creating the tools
  • Bottleneck to control access

to dangerous DNA

www.sechintower.com

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The Plan

Recycling Methods:

  • Minicolumns
  • Gels

DIY Methods:

  • Minicolumns
  • Enzyme Purification

○ RTX & ELP ○ Gel Purification

  • Enzymes

○ EcoRI, M.EcoRI

blogs.nature.com

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DIY Cost Comparison

Protocol # Price Transformations 4 $8.25 Plating 4 $0.71 Overnights 4 $0.15 Digests 4 $0.80 Ligations 1 $6.40 Minipreps 4 $1.74 Gels 2 $2.70

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DIY - Homemade Minipreps

  • DIY Buffers
  • Minicolumn Recycling
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DIY Buffers

  • Homemade Buffer Efficiency

❖ Increased overall efficiency from ~50% to ~60%

Yield Efficiency QIAprep Spin Miniprep Kit 219.6 ng/uL

  • Re-suspension Buffer Only

189.1 ng/uL 86% Lysis Buffer Only 221.5 ng/uL 101 % Neutralization Buffer Only 102.8 ng/uL 47% Binding Wash Only 240.2 ng/uL 109 % Column Wash Only 239.5 ng/uL 109%

Klenchin (2010).

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Minicolumn Recycling

Yield New Minicolumn 175.8 ng/uL Recycled Minicolumn 178.7 ng/uL

100% Efficiency 0% Carryover

Nagadenahalli B. Siddappa. et.al. (2007).

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DIY Minicolumns & Gel Recycling

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DIY Enzymes

Protocol # Price Transformations 4 $8.25 Plating 4 $0.71 Overnights 4 $0.15 Digests 4 $0.80 Ligations 1 $6.40 Minipreps 4 $1.74 Gels 2 $2.70

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ELP (Elastin Like Protein)

  • Oligomeric repeats of Val-Pro-Gly-Xaa-Gly
  • Fused at ends of proteins and precipitates at elevated

temperatures and increased concentrations of NaCl.

Meyer (1999)

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RTX (Repeats in Toxin)

  • Polypeptide repeats
  • Undergoes conformational change upon binding to

Calcium which causes them to precipitate.

  • We submitted an RTX to the registry in freighburg

format. BBa_K1188002

Shur (2013)

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RTX Characterization

  • Fused RTX to GFP
  • Grew in culture
  • Lysed cells
  • Treated lysed cells

with various CaCl

  • Results show 10mM

concentrations of Ca are effective in precipitating tag when attached to GFP

0 mM 0.1mM 1 mM 10 mM 100 mM Cell Lys

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RTX

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Gel Purification with Color Tags

  • Agarose Gel
  • AmilCP and RFP
  • Quick
  • Easy to do
  • High purity
  • Decent yields
  • Utilizes only common lab

materials

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Characterization

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Results of Separation

20% Yield 34% Yield

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The Plan

ChlorR E X P

J23100 B0034

EcoRI S AmpR E X P

J23100 B0034

M. EcoRI S

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Making the Plasmids

  • Origin of sequence:

REBASE

▫ Codon optimized

  • Process

▫ Gibson Assembly ▫ PCR ▫ Digestion/ Ligation

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Unfortunately...

AmpR E X S P

J23100

B0034

ChlorR E X S P

J23100

B0034

M. EcoRI

X P S EcoRI X P S

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Summary

Protocol # Price Transformations 4 $8.25 Plating 4 $0.71 Overnights 4 $0.15 Digests 4 $0.80 Ligations 1 $6.40 Minipreps 4 $1.74 Gels 2 $2.70

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Outreach

High School Curriculum

  • HS science labs can range thousands per year
  • Key curriculum involves gel electrophoresis,

restriction digestion, ect.

  • Future iGEMers in peril!

How can DIY bio help?

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Outreach (cont.)

The iGEM Experience:Creating DIY, safe, low-cost biology for the mass through kits.

Transformation (Chemical) DNA Chemically Competent Cells Antibiotics Digestion Enzymes: E, P, X (DIY) 10x NEBuffer 2.1 DNA Gel Electrophoresis DNA Loading Dye DNA Ladder Agarose powder (Recycle) Sybr Safe

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Concluding Notes

Excerpt from “Biopunk, DIY Scientists Hack the Software of Life”:

"We reject the popular perception that science is only done in million-dollar University, government, or corporate labs; we assert that the right of freedom

  • f inquiry, to do research and pursue

understanding under one's own direction, is as fundamental a right as that of free speech or freedom of religion."

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Thanks to Our Sponsors!!

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Questions?