2018

GEBZE TECHNICAL
UNIVERSITY
Molecular Genetics Laboratory
Turkey

EBRU AKHARMAN / 142204026

25.04.2018
 COLONY SELECTION TECHNIQUES - BLUE/WHITE
SCREENING
AIM

Molecular cloning can be accomplished by specific methods from the prokaryotic or eukaryotic cell
genome to which the gene encoding an important product or protein synthesis belongs (usually by
restriction endonuclease enzymes), which is transferred to a recipient prokaryotic or eukaryotic cell by
combining with a carrier vector DNA, provides genetic expression in the recipient cell. Bacterial
transformation is a natural process in which cells take up foreign DNA from the environment at a low
frequency. After transformation, the cells may express the acquired genetic information, which may serve
as a source of genetic diversity and potentially provide benefits to the host (e.g., antibiotic resistance).
With the molecular cloning , the process of transformation was exploited and enhanced to introduce
recombinant plasmid DNA into bacterial strains that were made “competent,” or more permeable, for DNA
uptake.In this experiment, transforming bacterias are selected with blue-white selection method. If the
bacterias have blue color, they were not transformed with plasmid DNA. If the bacterais have white color,
they were transformed with plasmid DNA.

INTRODUCTION
Blue – White Selection:
Blue-white screening is a rapid and efficient technique for the identification of recombinant bacteria in vector
based molecular cloning experiments. It relies on the activity of β-galactosidase enzyme occurring in E.coli
JM109, which cleaves lactose into glucose and galactose. The selection method is termed as the insertional
deactivation which is based on the deactivation of lacZ gene in plasmid vector due to insertion of DNA insert
(gene of interest) in MCS site within lacZ gene. It also requires use of specific host bacteria like E.coli JM109
with lacZ mutation, chromogenic compound X-Gal an analog of lactose and an inducer IPTG.

Lactose present in surrounding environment triggers the lacZ operon in E.coli JM109 to produce β-
galactosidase. Most plasmid vectors such as pUC19 carry a short segment of lacZ gene that contains coding
information for the first 146 amino acids of β-galactosisdase. The host E.coli JM109 strains used are
competent cells containing lacZ deletion mutation. When the plasmid vector is taken up by such cells, due to
α-complementation process, a functional β-galatosidase enzyme is produced.

The plasmid vectors used in cloning are manipulated in a way that this α-complementation process serves as a
marker for recombination. A multiple cloning site (MCS) is present within the lacZ sequence in the plasmid
vector. This sequence as a name suggests can be nicked by restriction enzymes to insert foreign DNA. When a
plasmid vector containing foreign DNA is taken up by host E.coli JM109, the α-complementation is not
permitted and functional β-galactosidase enzyme is not produced. On the contrary, if foreign DNA is inserted
at a location other than MCS or not inserted into the vector, the lacZ gene in plasmid vector complements lacZ
deletion mutation (referred as alpha complementation) in host E.coli JM109 producing a functional enzyme.
 Image 1: Structure of β-galactosidase

Assembly of β-galactosidase protein from wild type LacZ (left) and deletion mutant LacZ∆M15 (right). Deleting
only a few amino acids from the N-terminus of the protein results in an improperly assembled enzyme (dimer
rather than tetramer) that cannot catalyze the breakdown of lactose.

Image 2: X-gal Selection

For screening the clones containing recombinant DNA, a chromogenic substrate known as X-gal is added to
the agar plate. If β-galactosidase is produced, X-gal is hydrolyzed to form 5-bromo-4-chloro-indoxyl, which
spontaneously dimerizes to produce an insoluble blue pigment called 5,5’-dibromo-4,4’-dichloro-indigo. The
colonies formed by non-recombinant cells, therefore appear blue in color while the recombinant ones appear
white. The desired recombinant colonies can be easily picked and cultured.

Isopropyl β-D-1-thiogalactopyranoside (IPTG) is used along with X-gal for blue-white screening. IPTG is a non-
metabolizable analog of galactose that induces the expression of lacZ gene. It should be noted that IPTG is not
a substrate for β-galactosidase but only an inducer. For visual screening purposes, chromogenic substrate like
X-gal is required.

MATERIALS
 Solutions Required
 LB
 Ampicillin ( 10 mg/ml )
 X-gal solution ( 20 mg/ml )
 IPTG ( 100 mM )
 Preparing the Solutions
X-gal solution ( 20 mg/ml ): Dissolve X-gal in an eppendorf tube to a final concentration of 20
mg/ml in 100 % DMF (dimethylformamid). Store the stock solution at –20 °C in the dark. Discard the
stock solution if the color changes significantly. X-gal should be stored immediately upon receipt at –
20 °C.
IPTG ( 100 mM ): Dissolve 0,238 g of IPTG in 8 mL of 𝑑𝐻2 𝑂. Filter sterilize with a 0.2 μm syringe filter.
Store in 1 mL aliquots at -20 °C.

METHOD
 Pour sterile warm LB-agar containing 100 μg/ml ampicillin into two petri dishes(Do not add ampicillin
when your agar is extremly hot!).
 Dry LB plates in laminar flow for about 20 minutes.
 Mix 20 μl IPTG (100 mM) and 20 μl X-gal (20 mg/ml) with 60 μl LB in an eppendorf tube. Then put this
solution onto an agar plate.
 Spread evenly on the plate with a steril glass spreader.
 Dry the plate in the laminar flow until all the fluid has disappeared.
 Attach a numbered grid on the back side of each petri plate.
 Inoculate the plates with the bacteria that are to be tested by the method of toothpicking. For this;
pick each colony in the transformation plate with a sterile toothpick, and patch it on an LB-amp plate
first, and then on the second LB-amp plate containing X-gal and IPTG. (Only use each toothpick once.
Save the used toothpicks to be reautoclaved.)
 Incubate the plates overnight (12 to 16 hr) at 37 °C.
 Next day morning, incubate the plates at 4 °C for several hours. This allows the blue color to develop
fully.
 Examine the plate to determine the blue and white colonies and note the number of the white
colonies.
 RESULTS
Image 3: LB + AMP.
Randomly selected colonies from master petri dish are
inoculated primarily to LB + Ampicillin. Then, these
colonies are inoculated to LB + Ampicillin + Xgal +
IPTG. According to image 3 and 4, all colonies have
ampicillin resistant gene.
Image 5: Xgal + IPTG + LB + AMP.
According to image 5 and 6, all colonies have
ampicillin resistant gene but only some colonies have
Lacz gene. The blue colonies have to all of Lacz gene.
The white colonies do not have to all of Lacz gene.
Image 4: LB + AMP.
Randomly selected colonies from master petri dish
are inoculated primarily to LB + Ampicillin. Then,
these colonies are inoculated to LB + Ampicillin + Xgal
+ IPTG. According to image 3 and 4, all colonies have
ampicillin resistant gene.
Image 6: Xgal + IPTG + LB + AMP.
According to image 5 and 6, all colonies have
ampicillin resistant gene but only some colonies have
Lacz gene. The blue colonies have to all of Lacz gene.
The white colonies do not have to all of Lacz gene.
 DISCUSSION
The blue-white screen is a screening technique that allows for the detection of successful ligations in vector-
based gene cloning. DNA of interest is ligated into a vector. The vector is then transformed into competent
bacterial cells. The competent cells are grown in the presence of X-gal. If ligation was successful, the bacterial
colony will be white; if not, the colony will be blue. This technique allows for the quick and easy detection of
successful ligation. X-gal (5-bromo-4-chloro-indolyl-β-D-galactopyranoside) is an organic compound consisting
of galactose linked to a substituted indole. X-gal is much used in molecular biology to test for the presence of
an enzyme, β-galactosidase. X-gal is one of many indoxyl glycosides and esters that yield insoluble blue
compounds similar to indigo as a result of enzyme-catalyzed hydrolysis in this case by β-galactosidase. β-
galactosidase is a protein encoded by the lacZ gene of the lac operon, and it exists as a homotetramer in its
active state. Although the formation of colonies in the negative control group, recombinant colonies were
found in this experiment. Blue-white screening only indicates the presence of insert. Any cloning artifact that
disrupts the α-peptide DNA will also lead to a white colony. This situation is cause to wrongly positive results.
If a small fragment is inserted in-frame, read-through can lead to a functional β-galactosidase enzyme and a
blue colony. This situation is cause to wrongly negative results. Blue-white screening is just that – a screening
process. It does not select only those cells that have taken up a plasmid and thus should be used in
conjunction with selection methods. There are 9 colonies between 9-24 and 34-42, 3 colonies between 70-72,
12 colonies between 97-108, 6 colonies 115-120 in the Xgal+IPTG+LB+Ampicillin. Combining selection and
screening ensures that the white colonies you see are white due to successful cloning and not because the cell
failed to take up the α-complementation plasmid. This way you can quickly and easily identify colonies that
not only have your plasmid, but also confirm those plasmids have your insert.

RESOURCES

http://blog.addgene.org/plasmids-101-blue-white-screening

www.colby.edu/biology/BI279/BactTransf_S11.pdf

www.nhm.ac.uk/.../blue-white-colony-screening_aug12-11

www.csus.edu/indiv/r/rogersa/bio181/bluescript.pdf