Perform bacterial Transformation by standard method

Aim: To prepare competent cells, perform transformation and to learn the concept of α-
complementation.

Principle: Bacterial transformation is a process in which bacteria manage to uptake or

bring in free/external DNA from the environment/surrounding medium. This is a very basic
technique that is routinely used in a molecular biology lab. The purpose of this technique is
to introduce a foreign plasmid DNA into bacteria and to use these bacteria to amplify the
plasmid DNA. The ability to take up DNA efficiently by most bacteria is limited in nature.
However, bacterial cells can be artificially induced to take up DNA by treating them with
calcium chloride. Culture of such cells that are capable of taking up DNA is said to be
competent. The conditions required to produce competence vary from species to species.
The phenomenon of competence has not been understood very well. It appears to result
from changes in the cell wall of bacteria and is probably associated with the synthesis of
cell wall material at a particular stage of the life cycle of bacteria. In the course of
developing competence, receptors of some kind are either formed/activated on the cell
wall, which are responsible for initial binding of the DNA. The complex thus formed is
resistant to DNases. Cells are then exposed briefly to a temperature of 42 C ̊ heat shock,
wherein pores are created and DNA is taken up. Immediate chilling on ice ensures closure
of pores. Cells are then said to be transformed. These cells are then screened for
transformants/recombinants. Selection of cells containing foreign DNA is done based on
the selection marker carried by this DNA. Example, pUC plasmid has ampicillin resistance
factor that enables only transformed cells to grow on LB-Amp plates. Non-transformants,
which are ampicillin sensitive, do not produce colonies on the selective medium.
Transformants and non-transformants are therefore easily distinguished.

Identification of recombinants among the transformed cells is generally done by

insertional-inactivation. With most cloning vectors, insertion of DNA fragment into the
plasmid destroys the integrity of one of the genes present on the molecule. As a result, the
characteristic coded by the inactivated gene is no longer displayed by host cells and this is
called insertional-inactivation.
For example, pUC18 is a high copy number plasmid of size 2686 bp, with Col E1 origin of
replication. It carries a 54 bp polycloning region and ampicillin resistance marker, along
with coding information for the first 146 amino acids (amino terminal) of -galactosidase
(Lac Z) gene. Some strains of E. coli bear a deletion at the amino terminal end of Lac Z
gene and thus synthesize an inactive C-terminal fragment. On transforming such competent
bacterial strains with pUC18, the host and plasmid encoded fragments associate to form an
enzymatically active protein. This type of complementation is known as -complementation.
Lac+ bacteria that result from -complementation can be recognized as they form blue
coloured colonies in presence of X-gal (chromogenic substrate for -galactosidase) and
IPTG (inducer for the expression of the enzyme). However, insertion of a fragment of
foreign DNA into the polycloning site of plasmid results in production of an amino
terminal fragment that is not capable of-complementation. Hence, cells carrying
recombinant plasmid will form white colonies. This is also referred to as Blue-White
Screening.

Materials Required:
̊ shaker,
Equipment: Centrifuge (preferably refrigerated), Incubator, 37 C
Spectrophotometer
Glassware: Conical flask, Petri plates, Pipettes, Spreader
Reagent: LB broth, X-Gal, Ampicillin, 0.1M CaCl2, Plasmid DNA, E.coli DH5α, Distilled
water.
Other Requirements: Capped Centrifuge tubes, Crushed ice, Cuvette (of 1 cm path length),
Tips, Micropipette, Water bath.

Procedure:
Duration of experiment: Experiment has been carried out over a span of 3days,
approximate time taken on each day is indicated below:
Day 1: Media preparation, Revival of host – 2 hours
Day 2: Competent cell preparation and transformation – 6 hours
Day 3: Observation of transformants – 1 hour

Preparation of Competent Cells:

Day 1:
Media preparation:
Preparation of LB agar/broth (1 litre): 25g of media was dissolved in 800ml of distilled
water. The pH was adjusted to 7.0 with 5N NaOH and the volume was made upto 1000ml.
It was sterilized by autoclaving. LB agar can also be prepared by adding 1.5% agar and
then autoclaving.

Preparation of Antibiotic: 50mg of ampicillin was dissolved in 0.5ml of sterile water and
allowed to store at 4 ̊c. This was prepared just before use and was added only after the
medium was cooled enough.

Revival of Host: 1. The lyophilized vial was opened in the laminar flow hood.
2. The lyophilized sample was rehydrated by adding 0.1ml of LB broth.
3. A loopful of suspension was streaked onto LB agar plates and incubate at 37 ̊C
overnight.

Day 2:
Preparation of Competent Cells: 1. 3-5 moderately sized host colonies were picked up from
LB plate and was inoculated into 5ml LB broth.
2. It was incubated on a shaker at ~200 r.p.m at 37 C ̊ till the cells turn visibly turbid or OD
A600 reaches 0.23-0.26, this takes about 2-3 hours.
3. 1.5 ml of this culture was aseptically transferred into 2 sterile 1.5ml vials and was
spinned at 4000 rpm for 5 minutes at 4 ̊C.
4. The supernatant was drained completely by quickly tapping on tissue. It was
resuspended gently in 300μl of ice-cold 0.1M CaCl2 solution.
5. This was kept on ice for 15 minutes.
̊ .
6. Next it was kept on for spinning at 4000 r.p.m for 5 minutes at 4 C
7. The supernatant was again drained off and resuspended very gently in 100μl of ice-cold
0.1M CaCl2 solution. This made the competent cells now ready to use.

Transformation:
 2 μl of PUC18 DNA was added and labelled as A. The second vial was labelled B –
this was used as control with no DNA added.
 The vial A was tapped gently and now they (both vials) were incubated on ice for
15-20 minutes.
 An external heat shock was given by keeping the vials in water-bath adjusted to 42 ̊C
for precisely 2 minutes. Now immediately after 2 minutes they were shifted to chill
on ice for 10 minutes.
 1ml of LB was aseptically added to the vials and the cultures were incubated in a
shaker (horizontally using cellophane tape) for 1hour at 37 ̊C.

Plating:
 50 μl from vial A was plated on LB ampicillin plate and spreaded thoroughly using
spreader. It was labelled A.
 Next 100 μl from vial A was plated on LB ampicillin plate with 40 μl of X-Gal
thoroughly using spreader. It was labelled as X.
 100 μl from vial A was plated on LB without ampicillin. It was labelled as D.
 100 μl from vial B was plated on LB ampicillin plate similarly as above. It was
labelled B.
 100 μl from vial B was plated on LB without ampicillin. It was labelled as C.
 The plates were incubated overnight inverted at 37 C
̊ .

Day 3:

Observation: The plates were checked for observation the following day. The observation
according to plates have been tabulated. (To be drawn on white page beside observation’s
ruled page)

Specification A (+ DNA) B ( - DNA) C (- DNA) D ( + DNA) X (+ DNA)

Medium LB+Amp LB+Amp LB LB LB+Amp
Culture 50 μl 100 μl 100 μl 100 μl 100 μl
X-Gal - - - - 40 μl
White No colonies Lawn Lawn Blue
colonies- 8 colonies- 52
Calculation: (To be shown on white page beside observation’s ruled page)

Efficiency of the competent cells can be calculated as No. of transformants/ μg of DNA:

𝑐𝑓𝑢
𝑛𝑜.𝑜𝑓 𝑐𝑜𝑙𝑜𝑛𝑖𝑒𝑠 × 1000𝑛𝑔 𝑜𝑓 𝜇𝑔 𝑜𝑓 𝐷𝑁𝐴 𝑡𝑟𝑎𝑛𝑠𝑓𝑜𝑟𝑚𝑒𝑑
Transformation efficiency=
𝐴𝑚𝑜𝑢𝑛𝑡 𝑜𝑓 𝐷𝑁𝐴 𝑝𝑙𝑎𝑡𝑒𝑑

Amount of DNA transformed = 10 ng

For plate X:

Volume of culture plated = 100 μl

10 ×100
So, amount of DNA plated = ng = 1ng
1000

52 ×1000 𝑛𝑔
For 52 blue colonies, efficiency = = 5.2 × 104
1 𝑛𝑔

For plate A:

Volume of culture plated = 50 μl

10 ×50
So, amount of DNA plated = ng = 0.5 ng
1000

8 ×1000 𝑛𝑔
For 8 white colonies, efficiency = = 1.6 × 104
0.5 𝑛𝑔