Making and storing competent cells on a budget

All modern biotechnology labs need to make and test many different functional DNA molecules, but DNA in a test tube is little more than a heterogeneous water soluble polymer.  To unlock the power of functional DNA it needs to be inserted into a living cell.  Once the DNA is inside of a cell the molecular machinery of the cell decodes the information stored in the DNA and remarkable things can happen.  

These are agar plates with a selectable marker called ampicillin.  These plates can be used to identify E. coli cells that have taken up functional DNA.

The process of inserting DNA into a cell is referred to as transformation or transfection.  Most cells can be treated so they uptake some DNA, but only a few organisms, like yeast and E. coli, are especially well suited for DNA uptake.  At the Great Lakes Biotech Academy we are developing efficient ways to assemble and characterize functional DNA at a very low cost.  We have made significant progress on the DNA assembly portion (check for a future article on this topic), but we also need new low cost procedures for preparing and storing cells that can uptake DNA.  

These cells are blue because they were transformed with functional DNA that allows the cells to change color. 

Cells that take up DNA efficiently are called competent cells.  Typically competent cells are evaluated based on a metric called transformation efficiency.  Competent cells produced by commercial vendors have transformation efficiencies in the range of 10^8 or 10^9 colony forming units per microgram of DNA.  Translated to common English, that means if you mix DNA of a mass 1/100th the mass of a grain of salt with these commercial cells you will have approximately 1 billion cells uptake the DNA.  These commercial cells are a tremendous asset for biotech labs, but they are also very expensive.  Each use of competent cells from a typical vendor costs >$20.  The process of making cells competent involves treatments that make the cells very fragile and considerable care must be taken when handling them.  Because the cells are so sensitive they must be stored in special lab freezers that are kept at -80 degrees Celsius or lower (-112 Fahrenheit!).  The least expensive of these -80C freezers costs several thousand dollars.  This is way too expensive.

To fix this problem I performed a few simple experiments.  In the first experiment, I evaluated whether or not commercial vendor competent cells could be stored in a standard freezer at only -20 Celsius.  Unfortunately, the transformation efficiency of the cells was severely compromised by storage at this temperature.  Buying a $10,000 freezer was out of the question, so I next performed an experiment where I measured dry ice sublimation rates inside of a styrofoam cooler inside of a low cost chest freezer.  The chest freezer was only $60 and could easily hold temperature at -20C.  I figured that by keeping the dry ice inside of a cooler in a chest freezer it could be maintained for a much greater time.  I want to point out that dry ice is extremely cold (-78 Celsius) and should not be stored in direct contact with the inside walls of a refrigerator or freezer or inside of a sealed container.  The graph below shows the measured sublimation rate for the dry ice.  Under these conditions the dry ice sublimated at a rate of approximately 1.0-1.2 lbs per day.  Dry ice is available from grocery stores and commercial ice vendors for ~$1.60 per lb in central Indiana.  That works out to ~$50-60 per month to keep competent cells at close to -80C.  That is a significant expense, but dramatically cheaper than the deep freezer.  

Graph showing the dry ice sublimation rate under the conditions described in the post.

Graph showing the dry ice sublimation rate under the conditions described in the post.

By keeping the cells on dry ice and using a procedure to make our own competent cells we can evaluate functional DNA at a low cost.  The protocol to make the cells is time consuming, but several hundred aliquots can be made in a single day.  Using these approaches brings our total per transformation cost to less than $1 so long as we are doing several transformations per day.