Blue White Screening Protocol

Blue / White colony screening is a strategy to quickly and easily distinguish between recombinant and non-recombinant colonies. It requires a special vector and a special strain of E. coli. It is particularly helpful in tricky cloning strategies such as blunt ended cloning or DNA library preparation.

How Blue White Screening Works:

The first gene in the E. coli lac operon is lacZ, which encodes β-galactosidase (β-gal). The active form of β-gal is a tetramer and hydrolyses lactose into glucose and galactose. Deleting amino acids 11-41 of β-gal (called the lacZΔM15 mutation) means the enzyme is unable to form a tetramer and is non-functional (Langley et al. 1975). It was discovered that supplying amino acids 1-59 (the α-peptide) of β-gal in trans (separately) allowed the truncated β-gal to form tetramers and function again (Ullmann et al. 1967; Langley et al. 1975). Rescuing β-gal by supplying the α-peptide in this way was termed α-complementation. Later, Vieira and colleagues (Vieira & Messing 1982) realised that α-complementation could be used to screen E. coli colonies for the presence of inserts. They cloned the α-peptide coding region into a pUC plasmid and then introduced a multiple cloning site (MCS) into the middle of that region. When a piece of DNA is ligated into the MCS, it disrupts the α-peptide, rendering the β-gal non-functional.

5-bromo-4-chloro-indolyl-β-D-galactopyranoside (x-gal) is a colourless analogue of lactose. When β-galactosidase hydrolyses x-gal, it creates a blue product (5,5'-dibromo-4,4'-dichloro-indigo). In blue white screening, an E. coli strain is transformed with a ligation reaction and spread onto agar plates containing x-gal. A blue coloured colony indicates that the α-peptide in the plasmid is intact (no insert) whereas a white colony indicates that the α-peptide is disrupted (insert present).

What you will need for Blue White Screening:

Competent cells of an E. coli strain with the lacZΔM15 mutation. Common Blue white compatible strains include: XL1-Blue, XL2-Blue, DH5α F', DH10B, JM101, JM109 and STBL4. Please see the 'Competent cells' protocol for details about preparing E. coli cells for transformation.

A vector with the α-peptide coding region and MCS. Common blue white compatible vectors include: pGEM-T, pBluescript, pUC18 and pUC19 (see button below).

Your ligation reaction (i.e. your insert of choice into a blue white compatible vector, above).

Control plasmid (e.g. pBluescript).

Antibiotic for selecting for the vector.

X-gal 20 mg/ml. X-gal can be purchased ready dissolved or as a powder. It may be dissolved in DMSO or DMF at a concentration of 20 mg/ml. X-gal must be stored at -20°C and protected from light (by wrapping foil around the stock container).

Isopropyl β-D-1-thiogalactopyranoside (IPTG) 10 mM (needed to induce β-gal expression in the E.coli)

How to Prepare Blue White Screening Plates:

Prepare some LB agar plates containing the appropriate antibiotic to select for your chosen plasmid. Please see the 'Making agar plates' and 'Antibiotic concentrations' protocols.

Onto each plate to be used for blue white screening, spread:

  • 100 μl of 20 mg/ml x-gal stock and
  • 100 μl of 10 mM IPTG

and allow the plates to dry with the lid slightly open before use. This can be performed either next to a bunsen burner on the bench or in a laminar flow hood. Using a hood may dry out the plates if they are left for too long. 

Bacterial Transformation:

Transform your ligation reaction(s) into competent E. coli cells as usual. Spread the transformation reaction onto an x-gal IPTG plate (prepared as above). Incubate the plate overnight at 37°C. Once the colonies have grown, the plate may be incubated at 4°C for 1 hour. This helps the blue colour to develop making it easier to discern the negative colonies.

It is a good idea to include a control. Transform an aliquot of E. coli with an intact α-peptide-containing-plasmid (pUC19 for instance, see button below). The colonies on this control plate should all be blue. If they're not, then the x-gal may not have been spread evenly or the antibiotic may not be working properly.

Some notes:

This screen does not give any information about the direction of an insert, just its presence or absence. If the insert is quite short and maintains the frame of the α-peptide, it is possible (but unlikely) that it will produce a functional α-peptide fusion giving blue colonies even when the insert is there (false negatives). However, these colonies will likely be a lighter blue than the true negatives. It is also possible (but again unlikely) to get a white colony with no insert (false positive). This could result from nuclease degradation of the linearised vector disrupting the α-peptide before re-ligation. Therefore, it is always a good idea check your insert by sequencing too.

 

 

Blue White Screening References:

Langley, K.E. et al., 1975. Molecular basis of beta-galactosidase alpha-complementation. Proceedings of the National Academy of Sciences of the United States of America, 72(4), pp.1254–1257.

Ullmann, A., Jacob, F. & Monod, J., 1967. Characterization by in vitro complementation of a peptide corresponding to an operator-proximal segment of the beta-galactosidase structural gene of Escherichia coli. Journal of molecular biology, 24(2), pp.339–343.

Vieira, J. & Messing, J., 1982. The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene, 19(3), pp.259–268.

 

Author: Dr Richard Parker-Manuel and Dr Ryan Cawood