Bacterial Transformation Protocol with Competent Cells

Bacterial Transformation using Competent Cells: Summary

Since we have already learned Calcium Phosphate Transfection with mammalian cells, let’s now focus on bacterial transformation of DNA with competent cells. In general, bacterial cells take up naked DNA molecules or plasmids via a process called transformation. Usually, this happens at a slow rate, but when bacterial cells die in close proximity to others, or when they are stressed, the transformation process occurs at a much higher rate. However, not all bacterial cells can be transformed, so biologists use ‘Competent Cells’ which are more inclined to take up DNA. The end goal of transformation is to get bacteria that have your genes of interest so that they will replicate your genes along with their own. If the bacteria contain your genes of interest, you can use them to mass produce proteins, or just store them for extended periods of time because bacteria are so hardy. A good way to test whether your genes of interest were transformed is to include antibiotic resistance in your plasmid. This way, you can be fairly certain that if your bacteria are resistant to antibiotics, they are also carrying genes of interest to you.

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Take a look at how natural transformation works:

Transformation Biology in Bacteria

For bacteria, survival is key and transformation is one of their survival mechanisms. As biologists, we can make use of this survival mechanism for our benefit as well. To do this, we first incubate our competent bacteria with our plasmid and calcium chloride. Bacterial membranes are permeable to chloride ions, but not to calcium. So, as chloride ions enter the cell, the bacteria tend to swell (because they also intake water with chloride ions). Then we heat the bacteria in a process called ‘heat shock’ such that they turn on their survival genes. This causes the bacteria to uptake the surrounding plasmids. With the right design, this plasmid will then be recognized by bacterial DNA polymerases (remember our PCR Guide ?) and it will be expressed/replicated along with the bacteria’s normal DNA.

Take a look below to understand how biologists transform cells:

Selecting for Transformed Bacteria with the Lac Z Operon

Once your target plasmid is inside the bacteria, you still need to separate transformed cells from those that are not transformed. Another key challenge is that the transformation process may lead to some DNA being recombined so that your gene of interest is no longer functional. How do you select for cells that only contain functional target DNA that hasn’t been recombined? The trick is to use both antibiotic resistance and a Lac Z operon. By cloning your plasmid along with a Lac Z operon, you give your cells the ability to make a galactosidase protein. If cells have the galactosidase and you feed them X-Gal, they turn blue; cells without this operon are white. So, you first transform all your cells. Then you feed them IPTG to activate the Lac Z operon and cause cells to produce the galactosidase. Then you add in X-Gal and just pick out the bacteria that have functional Lac Z because the useful cells will be a bright blue!

Check out the figure below:

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Bacterial Transformation Protocol

Transformation describes the uptake and incorporation of plasmid DNA into bacteria. Antibiotic resistance genes carried on plasmids allow selection of transformants. This protocol describes the transformation of DH5α E. coli with pAdtrackCMV (a vector carrying kanamycin resistance).

Materials for Bacterial Transformation

Ligation mix (20 µl) – insert ligated into pAdTrack-CMV shuttle vector (Plasmid #16405, Addgene)
DH5α competent cells (includes pUC19 DNA control; #18265017, ThermoFisher Scientific)
LB broth (#10855-021, ThermoFisher Scientific)
LB Agar selective plates (prepare from #22700025, ThermoFisher Scientific) with 50 µg/ml kanamycin (#15160054, ThermoFisher Scientific)

Step by Step Transformation Protocol

1. Thaw competent cells on ice. Aliquot 50 µl into cooled Eppendorf tubes for each transformation reaction.*
2. Add 5 µl of ligation mix to each tube.*
3. Incubate on ice for 30 min.
4. Heat-shock the cells for 20 sec in a 42°C waterbath.
5. Place on ice for 2 min.
6. Add 950 µl of warm LB broth per tube.
7. Allow cells to recover at 37°C for 1 hour with gentle shaking.
8. Spread 200 µl and 20 µl of each transformation mix onto warm selective plates.*
9. Incubate plates overnight at 37°C.

 

Notes on this methodology

• We will talk about “Ligation” in another future blog post
• Step 1. Unused cells can be refrozen and stored at -80°C for future use.
• Step 2. As a transformation control, add 1 µl of pUC19 plasmid to one aliquot of cells (pUC19 confers resistance to ampicillin so will need to be seeded onto different selective plates).
• Step 8. Transformation mix can be stored at 4°C and plated the next day if required.

Bacterial Transformation Video Tutorial

Applications of DNA Transformation on Scigine

• Bacterial Transformation with pORFES plasmid
• Natural Transformation of Competent Cells
• PCR Followed by Transformation and Cloning
• Sequencing Hamster Testes using RT-PCR and Bacterial Transformation
• Fusion Protein Transformed into BL21

References

Excellent Book about Bacterial Transformation
Guide to Common terms in Transformation – Oklahoma University
Compilation of History of Transformation and related protocols
He et al Proc Natl Acad Sci U S A. 1998 Mar 3. 95(5):2509-14.