Introduction to Blue-White Screening – Background and Protocols for Colony Selection

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Introduction

Blue-white screening is a rapid and efficient technique for the identification of recombinant bacteria. It relies on the activity of β-galactosidase, an enzyme occurring in E. coli, which cleaves lactose into glucose and galactose.

Background

The presence of lactose in the surrounding environment triggers the lacZ operon in E. coli. The operon activity results in the production of β-galactoisdase enzyme that metabolizes the lactose. Most plasmid vectors carry a short segment of lacZ gene that contains coding information for the first 146 amino acids of β-galactosisdase. The host E. coli strains used are competent cells containing lacZΔM15 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 such 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 can be nicked by restriction enzymes to insert the foreign DNA. When a plasmid vector containing foreign DNA is taken up by the host E. coli, the α-complementation does not occur, therefore, a functional β-galactosidase enzyme is not produced. If the foreign DNA is not inserted into the vector or if it is inserted at a location other than MCS, the lacZ gene in the plasmid vector complements the lacZ deletion mutation in the host E. coli producing a functional enzyme.

Principle

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.

A schematic representation of a typical plasmid vector that can be used for blue-white screening

Figure 1: A schematic representation of a typical plasmid vector that can be used for blue-white screening.

A schematic representation of a typical blue-white screening procedure

Figure 2: A schematic representation of a typical blue-white screening procedure.

Blue-white screening products offered by Sigma-Aldrich

Product No. Name Solubility Action Application Special features
B6650 X-GlcA DMF Substrate for ß-galactosidase and ß-glucuronidase gene detection screening of recombinant bacteria • blue-white screening
16658 X-GalNAc DMF, DMSO Substrate for N-acetyl-ß-galactosidase screening of recombinant bacteria and Candida albicans • blue-white screening
S7313 S-Gal® sodium salt Water Substrate for ß-galactosidase gene detection screening of recombinant bacteria • blue-white screening
• autoclavable
• enhanced contrast for automated colony counting
S9811 S-Gal® Water Substrate for ß-galactosidase screening of recombinant bacteria • blue-white screening
• autoclavable
• enhanced contrast for automated colony counting
C4478 S-Gal®/LB Agar Blend Water Substrate for ß-galactosidase screening of recombinant bacteria • blue-white screening
• autoclavable
• enhanced contrast for automated colony counting
• has added IPTG
B2904 Bluo-Gal DMF Substrate for ß-galactosidase screening of recombinant bacteria • blue-white screening
• no sterilization required
B3928 Blue-White Select™ Screening Reagent Ready-to-use solution Substrate for ß-galactosidase screening of recombinant bacteria • blue-white screening
16669 Magenta-Gal DMF, DMSO Substrate for ß-galactosidase screening of recombinant bacteria • red-white screening
B8931 Magenta-Gal Methanol Substrate for ß-galactosidase screening of recombinant bacteria • red-white screening
B4252 5-Bromo-4-chloro-3-indolyl ß-D-galactopyranoside DMF, DMSO Substrate for ß-galactosidase screening of recombinant bacteria • blue-white screening
• can be used for immunocytochemistry
• no sterilization required
B9146 5-Bromo-4-chloro-3-indolyl ß-D-galactopyranoside DMF, DMSO Substrate for ß-galactosidase screening of recombinant bacteria • blue-white screening
• no sterilization required
B6024 5-Bromo-4-chloro-3-indolyl ß-D-galactopyranoside (tablet) DMF, DMSO Substrate for ß-galactosidase screening of recombinant bacteria • blue-white screening
• no sterilization required
16665 5-Bromo-4-chloro-3-indolyl ß-D-galactopyranoside DMF, DMSO Substrate for ß-galactosidase screening of recombinant bacteria • blue-white screening
• no sterilization required
I1284 Isopropyl ß-D-thiogalactopyranoside solution Ready-to-use solution Induces Lac promoter screening of recombinant bacteria • blue-white screening
• can be added to culture media
I6758 Isopropyl ß-D-1-thiogalactopyranoside Water Induces Lac promoter screening of recombinant bacteria • blue-white screening
I5502 Isopropyl ß-D-1-thiogalactopyranoside Water Induces Lac promoter screening of recombinant bacteria • blue-white screening

Protocols

The complete protocol of blue-white screening includes 3 important steps:

  • Ligation: ligation of foreign DNA into MCS of the plasmid vector
  • Transformation: introduction of plasmid vector with foreign DNA insert into competent E. coli
  • Screening: blue-white screening to identify recombinant bacterial colonies

Ligation

Sigma-Aldrich offers the following ready-to-use expression vectors that can be used for stable as well as transient expression systems. These FLAG-fusion constructs have origins of replication for propagation in both bacterial and mammalian cells.

 

Product No. Name Compatibility
E6783 pFLAG-CMV™-3 Expression Vector Bacteria, mammalian cells
E7658 p3XFLAG-CMV™-10 Expression Vector Bacteria, mammalian cells
E7283 p3XFLAG-myc-CMV™-26 Expression Vector Bacteria, mammalian cells
E9033 pFLAG-Myc-CMV™-22 Expression Vector Bacteria, mammalian cells
E6908 pFLAG-CMV™-5.1 Expression Vector Bacteria, mammalian cells
E9408 p3xFLAG-Myc-CMV™-25 Expression Vector Bacteria, mammalian cells
FLMAS Mammalian Amino-terminal FLAG Stable Expression Kit Bacteria, mammalian cells
E8158 pFLAG-ATS™ Expression Vector Bacteria
E8408 pFLAG-CTC™ Expression Vector Bacteria
D3404 pUC19 plasmid DNA from E. coli RRI Bacteria
D4154 pUC18 plasmid DNA from E. coli RRI Bacteria


Protocol

Materials required

 

Reagents Equipment
T4 DNA ligase buffer (KEM0049B) PCR reaction tubes
Plasmid vector Thermo cycler (optional)
Insert (foreign DNA) PCR clean-up column (NA1020)
T4 DNA ligase (KEM0020, KEM0019)  
Sterile, nuclease-free water  


The following is the reaction setup for ligation

 

Component                      Volume (µL)                  Final concentration
T4 DNA ligase buffer 2 1X
Vector x 1-10 ng/µL
Insert x 1-10 ng/µL
T4 DNA ligase 1 6U/µL
Sterile water x NA
Total volume 20  

 

  • Add all the above components into a clean reaction tube.
  • Incubate for 30 minutes at 25°C (can be performed in a thermo cycler).
  • Purify DNA using PCR clean-up column and elute in approximately 50 µL.
  • Transform 0.1-10 ng of the ligation product into chemical or electrocompetent cells that are compatible with the vector.

 

Transformation

High quality plasmid is essential for the transformation procedure. The following are the kits offered by Sigma-Aldrich to isolate and purify high quality plasmid suitable for transformation.

 

Product No. Name
PLN10 GenElute™ Plasmid Miniprep Kit (10 preps)
PLN70 GenElute™ Plasmid Miniprep Kit (70 preps)
PLN350 GenElute™ Plasmid Miniprep Kit (350 preps)
PFM10 GenElute™ 5 Minute Plasmid Miniprep Kit (10 preps)
PFM50 GenElute™ 5 Minute Plasmid Miniprep Kit (50 preps)
PFM250 GenElute™ 5 Minute Plasmid Miniprep Kit (250 preps)
PLX15 GenElute™ Plasmid Maxiprep Kit (15 preps)
PLD35 GenElute™ Plasmid Miniprep Kit (35 preps)


The detailed protocol for transformation using chemical or electrocompetent cells can be found here.

 

Screening

Sigma-Aldrich offers a range of chromogenic substrates that aid screening of recombinant bacteria. Some products may be used to spread on LB agar plates (screening protocol 1), while the others are incorporated into the microbial medium (screening protocol 2). The products are used along with IPTG wherever required. The protocols for both the procedures are given below.

Screening protocol 1 (applicable for product numbers B6650, 16658, B2904, B3928, 16669, B8931)

  • Spread 40 µL or appropriate amount of stock solution of chromogenic substrate and 10 µL of IPTG solution on LB agar plates using a sterile spreader (addition of IPTG when using product number B3928 is not required as it already contains IPTG).
  • The plates should include those with appropriate antibiotic and without antibiotic as controls.
  • Leave the plates to dry in laminar flow chamber with lids slightly open.
  • Spread 10-100 µL of transformed E. coli cells onto the LB agar plates using sterile spreader.
  • Incubate the plates at 37°C for 24-48 hours.
  • Blue and white colonies appear on the agar surface. Select the recombinant cells in the white colonies to culture.

Screening protocol 2 (applicable for product numbers S7313, S9811, C4478)

  • Prepare LB agar by weighing appropriate powder medium, agar and water in a sterile flask. Alternatively, weigh appropriate amount of C4478 and add to water in a sterile flask.
  • Add 300 mg/L of product numbers S7313 and S9811, and 500 mg/L of ferric ammonium citrate (product number F5879) to the medium before autoclaving. Avoid this step if C4478 is being used.
  • Autoclave the medium and cool just enough to be able to handle the flask.
  • Add appropriate concentration of selected antibiotic to the medium.
  • Pour approximately 25-30 mL of the LB agar into sterile plates and allow to set with lids slightly open.
  • Spread 10-100 µL of transformed E. coli cells onto the LB agar plates using sterile spreader.
  • Incubate the plates at 37°C for 24-48 hours.
  • Blue and white colonies appear on the agar surface. Select the recombinant cells in the white colonies to culture.
Blue-white color selection of recombinant bacteria using X-gal

Figure 3: Blue-white color selection of recombinant bacteria using X-gal.

Limitations of blue-white screening

  • The blue-white technique is only a screening procedure; it is not a selection technique.
  • The lacZ gene in the vector may sometimes be non-functional and may not produce β-galactosidase. The resulting colony will not be recombinant but will appear white.
  • Even if a small sequence of foreign DNA may be inserted into MCS and change the reading frame of lacZ gene. This results in false positive white colonies.
  • Small inserts within the reading frame of lacZ may produce ambiguous light blue colonies as β-galactosidase is only partially inactivated.

References

Molecular cloning: a laboratory manual. Sambrook, J., Fritsch, E.F., and Maniatis, T., New York: Cold spring harbor laboratory press, 1989.
Screening of Bacterial Recombinants: Strategies and Preventing False Positives, Padmanabhan, S., Banerjee, S., Mandi, N. (2011). Molecular Cloning - Selected Applications in Medicine and Biology, Prof. Gregory Brown (Ed.), ISBN: 978-953-307-398-9, InTech.