Team:Sydney Australia/Implementation

An Economic Comparison

What is the economic inequity that we're really tackling here? What are the overt and hidden costs associated with synthetic biology and the transformation of E. coli that act as barriers to entry? We had a look at our existing transformation protocols to try to get some ideas.

The main expenses that we identified were the capital expenses associated with acquiring all the necessary equipment. A very bottom of the line electroporator will set you back at least $3000 AUD, and -80°C freezers can cost an order of magnitude on top of that. Also, bear in mind that this is purely the purchasing cost in a developed country (Australia) with robust supply lines and local scientific instrument manufacturing. The delivered and installed costs would be prohibitive in other parts of the world, and prohibitive to educational institutions who need to prioritise costs across their whole curricula. Add onto this ongoing utility costs such as electricity for freezers and you have an unsustainable and infeasible investment.

The chemical method is not ideal either. The active ingredient (rubidium chloride) is only available in bulk, and costs upwards of $500 AUD per purchase, and each time the solutions required for transformation are made up, it is an immediate $50 investment. Not terrible as a one off, but rigorous science and teaching requires many runs to ensure reliability and acceptable results. The costs add up considerably quickly, leaving synthetic biology on the chopping block in comparison to other educational and research operations.

If we could cut out or attenuate these costs, it would open the door to many institutions offering synthetic biology and broadening the diversity and depth of experience in synthetic biology worldwide. We propose such strategy of our synthetic biology tool — Free coli that would be useful in a number of end users such as university researchers, manufacturers in biotechnology companies, but more importantly, to a wider range of the SynBio global community involving the brilliant minds in low-socioeconomic countries and areas that couldn't afford high-cost ab equipments. We would be giving them the power to freely participate in synthetic biology research and innovate the next big impactful SynBio tech-development!

What are the safety aspects we would need to consider?

Are we going to create the next superbug by genetic engineering/recombineering strategy? Well, this was definitely one of the many safety aspects that we avoided by designing our novel recombineering gene cluster insertion of replacement sequentially as well as inducible salicylate promoter controlling the expression levels of the Type-IV pilus assembly. Later sequential insertions will target the resistance gene of the previous insertion so that the strain does not build up an excessive number of antibiotic resistances. And, we are still able to select the chromosomal insertions at each stage. In addition, it would be difficult for this bacterium to be converted into a pathogen from its current status as a very safe lab model organism. JM109 and other E.coli strains used for cloning contain many intentional mutations and also many accumulated unintentional mutations that make them good at growing under lab conditions but also make them survive poorly in the environment or the human body. We also consider that the creation would first be checked and follow the guidelines within regulatory bodies as any novel SynBio Research would be assessed upon its safety profiles.

We envision others using our project with the help of our constructed Free coli protocol and the ease of having a laboratory math cheatsheet to circumvent the issues for conducting SynBio Research involving bacteria transformation of E.coli. We plan to implement this great SynBio Research tool by mainly two-phase approach: Despite the fact that we were unable to access the wet lab and validate our design due to Sydney's COVID-19 shutdown, we were able to produce many contributions to give to the iGEM community! Novel parts, our novel recombineering strategy for multiple gene cluster insertions ('Babushka Blocks,' our modelling of natural transformation proteins, and our theoretical design for a naturally transformable lab strain of E. coli, as well as educational resources to improve synthetic biology accessibility and inspire the next generation of synthetic biology researchers, are among them. We plan to test and optimise our design following on during Phase II of the project to carry out the validation and implementation.

Free Coli Transformation Protocol and Calculations Cheatsheet

  1. Thaw Free coli competent cells on ice.
  2. Chill approximately 5 ng (2 µl) of the ligation mixture in a 1.5 ml microcentrifuge tube.
  3. Add 50 µl of competent cells to the 5uL pure DNA (A260/A280~1.6). Mix gently by pipetting up and down or flicking the tube 4-5 times to mix the cells and DNA. Do not vortex.
  4. Place the mixture on ice for 30 minutes. Do not mix.
  5. Heat shock at 42°C for 30 seconds. Do not mix.
  6. Add 950 µl of room temperature media to the tube.
  7. Place tube at 37°C for 60 minutes. Shake vigorously (250 rpm) or rotate.
  8. Warm selection plates to 37°C.
  9. Spread 50-100 µl of the cells and ligation mixture onto the plates.
  10. Incubate overnight at 37°C.


  • Shaking incubator at 37°C
  • Stationary incubator at 37°C
  • Water bath at 42°C
  • Ice bucket filled with ice
  • Microcentrifuge tubes
  • Sterile spreading device


  • LB Agar Plate (with appropriate antibiotic)
  • LB or SOC media
  • Free coli cells
  • DNA you'd like to transform

Calculations for DNA and bacteria cell culture

Calculating Concentration of DNA & RNA by measuring OD260
For double-stranded DNA:
C = 50 x OD260 x df

For double-stranded DNA:
C = 33 x OD260 x df

C = Concentration of nucleic acid in µg/mL
OD260 = Absorbance at 260 nm
df = dilution factor (i.e., how much the nucleic acid in the cuvette was diluted)
If you took 10µl of your DNA/RNA and diluted it in 90µl of dilutant this would be a 1 in 10 dilution, and your dilution factor would be 10.

Any notable absorption above background for DNA and RNA at the below wavelengths indicates contamination:

Contaminant Wavelength
Organic compounds, thiocyanates and phenolate ions ~230
Phenol ~270
Protein ~280
Particulates >330

Calculating amount of insert for ligations
1:1 ratio of vector to insert: lw=(1/V) x Vw

I = length of insert in kb
V = length of vector in kb
Vw = vector weight in ng
Iw = insert weight in ng

1:X ratio of vector to insert: lw=((1/V) x Vw) x X
I = length of insert in kb
V = length of vector in kb
Vw = vector weight in ng
Iw = insert weight in ng
X = ratio of insert to vector (e.g., if a 1:3 ratio of vector to insert desired, X = 3)

Transformation efficiency (TE) equation
TE = Colonies/µg/Dilution
Colonies = the number of colonies counted on the plate
µg = the amount of DNA transformed expressed in µg
Dilution = the total dilution of the DNA before plating

TE calculation example: Transform 2 µl (100 pg) of control pUC19 DNA into 50 µl of cells, outgrow by adding 250 µl of SOC and dilute 10 µl up to 1 ml in SOC before plating 30 µl. If you count 150 colonies on the plate, the TE is:

Colonies = 150
µg DNA = 0.0001
Dilution = 10/300 x 30/1000 = 0.001
TE = 150/0.0001/0.001 = 1.5 x 109 cfu/µg