Practical 3A & 3B STB2163 Recombinant DNA Technology Semester 2, Session 2016/2017

PRACTICAL 3A:
THE INOUE METHOD FOR PREPARATION OF COMPETENT E. COLI
ULTRACOMPETENT CELLS.

Introduction:

Bacteria are able to take up DNA from their environment (exogenous DNA) in three ways;
conjugation, transformation, and transduction. Only transformation is the direct uptake of
DNA, since conjugation requires cell-cell contact via a sex pilus and transduction requires a
bacteriophage intermediary to transfer DNA from one cell to another.

For a bacterial cell to take up DNA from its surroundings, it must be in a special physiological
state called competence. Experiments by Frederich Griffith in 1929 using competent
Streptococcus (now Enterococcus) pneumoniae were instrumental in showing that DNA was
the transforming principle the genetic material.

Natural competence is highly regulated in bacteria, and the factors leading to competence
vary among genera. For some genera, only a portion of the population is competent at any
time; for others, the entire population gains competence. A series of competence proteins is
produced, which have some homology but differ in the Gram negative and the Gram
positive bacteria.

Artificial competence is not encoded in the cell's genes. Instead it is a laboratory procedure
in which cells are passively made permeable to DNA, using conditions that do not normally
occur in nature. These standard procedures are comparatively easy and simple, and can be
used to genetically engineer bacteria. Chilling bacterial cells in the presence of divalent
cations such as CaCl2 or MgCl2 prepares the bacterial cell walls to become permeable to
plasmid DNA. However, transformation efficiency is, in general, low. Only a portion of the
cells become competent and a fraction of those are successful in taking up DNA.

This Inoue protocol reproducibly generates competent cultures of E. coli that yield 1 x 108 to
3 x 108 transformed colonies/g of plasmid DNA. The protocol works optimally when the
bacterial culture is grown at 18C. If a suitable incubator is not available, a standard
bacterial shaker can be set up in a 4C cold room and regulated to 18C.

Objective:
The objectives of the experiments are to get the students to be familiarize with how cells
are made competent which is the primary step for transformation.

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Practical 3A & 3B STB2163 Recombinant DNA Technology Semester 2, Session 2016/2017

EXPERIMENT 3A
THE INOUE METHOD FOR PREPARATION OF COMPETENT E. COLI ULTRA
COMPETENT CELLS.

Materials:

Buffers & Solutions

1. High quality dimethylsulfoxide (DMSO).

2. 0.5M PIPES (piperazine1,2bis[2ethanesulfonic acid]) at pH 6.7.

Dissolve 15.1g of PIPES in 80mL MilliQ H2O. Adjust pH of the solution to 6.7 (with
KOH or HCl). Add MilliQ H2O to final volume of 100mL. (Optional: sterilize
solution by vacuum filtration in prerinsed Nalgene filter of 0.45m pore size).

3. Inoue Transformation Buffer (chill to 0C before use).

Dissolve solutes below in 800mL of MilliQ H2O.

Reagent Qty/L Final Concentration.

MnCl24H2O 10.88g 55mM
CaCl22H2O 2.20g 15mM
KCl 18.65g 250mM

Add 20mL PIPES (0.5M, pH 6.7). Adjust total volume to 1L with MilliQ H2O.
Sterilize Inoue transformation buffer by filtration through prerinsed 0.45m
Nalgene filter

Media
1. Luria bertani (LB) or Super Optimal Broth (SOB) for initial culture growth.

Centrifuge & Rotors

1. Centrifuge machine with rotor that fit 50mL Falcon tubes.

Special Equipments
1. Liquid nitrogen
2. Polypropylene tubes
3. Shaking incubator (18C)
4. Water bath at 42C
5. Chilled microfuge tubes

Reference
Inoue H., Nojima H., and Okayama H. (1990). High efficiency transformation of
Escherichia coli with plasmids. Gene, 96, 23-28.

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Practical 3A & 3B STB2163 Recombinant DNA Technology Semester 2, Session 2016/2017

Methods:

Step 1: Growing Bacterial Cultures

1. Pick a single bacterial colony (23 mm in diameter) from a plate that has been
incubated for 1620 hours at 37C.
2. Transfer colony into 25 mL of LB broth or SOB medium in at 250mL flask.
3. Incubate culture for 1620 hours at 37C with vigorous shaking (250300rpm).

Step 2: Harvesting Cells and Freezing Competent Cells

1. Inoculate a flask of 30 mL LB broth or SOB medium with 3 mL of culture from

Step 1 (starter culture).
2. Incubate flask at 37C with moderate shaking.
3. Read the OD600 of all three cultures. Continue to monitor every 15 mins until
reading is at 0.55.
4. Transfer the culture vessel to ice water bath for 10 min.
5. Harvest cells by centrifugation at 7500 rpm for 5 minutes at RT in 50 mL falcon
tube.
6. Pour off medium and dry the tube (inverted) on paper towels for 2min (use
vacuum aspirator to remove any drops of remaining medium adhering to walls of
the centrifuge bottle or trapped in its neck).
7. Resuspend the cells gently (by swirling) in 30 mL of icecold (0C) Inoue
transformation buffer.
8. Harvest cells by centrifugation at 7500 rpm for 5 minutes at room temperature.
9. Pour off the medium and dry the tube on paper towels for 2 min (use vacuum
aspirator to remove any drops of remaining medium adhering to walls of the
centrifuge bottle or trapped in its neck).
10. Resuspend cells GENTLY in 10 mL of Inoue transformation buffer (0 C).
11. Add 0.75 mL of DMSO (swirl to mix bacterial suspension).
12. Store on ice for 10 min.
13. Quickly dispense 50 L aliquots of suspensions into chilled, sterile microfuge
tubes. (10mL of suspension > 200 tubes of 50L).
14. Snap freeze competent cells in liquid nitrogen (store stock at 80C).
15. When needed, remove tube of competent cells from freezer, thaw in hand, use
immediately.

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Practical 3A & 3B STB2163 Recombinant DNA Technology Semester 2, Session 2016/2017

EXPERIMENT 3B
LIGATION OF THE PCR DNA FRAGMENT INTO CLONING VECTOR

Introduction:
Ligation of DNA is a central step in many modern molecular biology workflows. DNA ligation
is the process of joining together two DNA molecules ends (either from the same or
different molecules). Specifically, it involves creating a phosphodiester bond bond between
the 3' hydroxyl of one nucleotide and the 5' phosphate of another. This reaction is usually
catalyzed by a DNA ligase enzyme. This enzyme will ligate DNA fragments having blunt or
overhanging, complementary, 'sticky' ends. Typically, it is easier to ligate molecules with
complementary sticky ends than blunt ends. T4 DNA ligase is the most commonly used DNA
ligase for molecular biology techniques and can ligate 'sticky' or blunt ends.

The two components of the DNA in the ligation reaction should be equimolar and around
100g/ml. Most commonly, one wants to ligate an insert DNA molecule into a plasmid,
ready for bacterial transformation. Typically, DNA and plasmid vector are individually cut to
yield complementary ends, then both are added to a ligation reaction to be circularised by
DNA ligase. If the plasmid backbone to insert DNA ratio is too high then excess 'empty'
mono and polymeric plasmids will be generated. If the ratio is too low then the result may
be an excess of linear and circular homo- and heteropolymers.

The pGEM-T and pGEM-T Easy Vectors used are linearized cloning vectors with a single 3-
terminal thymidine at both ends. The T-overhangs at the insertion site greatly improve the
efficiency ofligation of PCR products by preventing recircularization of the vector and
providing a compatible overhang for PCR products generated by certain thermostable
polymerases. PCR product generated will have with 3A-tailed fragments that will
compliment the linearized cloning vectors with a single 3-terminal thymidine (T-overhangs)
at both ends.

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Practical 3A & 3B STB2163 Recombinant DNA Technology Semester 2, Session 2016/2017

Objective:
The objective of the experiments is to get the students to be familiar with the ligation of
DNA fragment or PCR product and formed cloned plasmid DNA for the subsequent
transformation experiments.

Materials: -
pGEM-T or pGEM-T Easy Vector with T-overhang system
pCR product with 3A-tailed fragments as insert DNA
T4 DNA ligase
Eppendorf tubes
Deionised water
Water bath

Method: -

1. Set up one ligation reaction per sample, plus one background, and one positive control.

2. Set up the following ligation reactions:

Reaction components Sample (+ve control) (-ve control)

2X rapid ligation buffer 5 mL 5 mL 5 mL

pGEM-T or pGEM-T Easy

Vector (50 ng) 1 ml 1 mL 1 mL

pCR product with

3A-tailed fragments 3 mL max - -

Control insert DNA - 2 mL -

T4 DNA Ligase (3 Weiss units/l) 1 mL 1 mL 1 mL

Deionised water - 1 mL 3 mL

3. Mix the reactions by gentle pipetting. Use 1.5 ml of autoclaved Eppendorf tubes for
ligations.

4. Keep in a refrigerator (4oC) and leave for overnight incubation.