Beta-galactosidase Plasmid Information

 

Beta-galactosidase features and uses:

 

 

Beta-galactosidase is a hydrolase enzyme that catalyzes the hydrolysis of ß-galactosides into monosaccharides. It is a commonly used and versatile reporter gene, suitable for producing an enzymatic colour change during histology and also for activating fluorescent prodrugs for visualisation in flow cytometry.


 

Beta-galactosidase also provides the basis for the blue/white screening of recombinant clones. This approach relies on the enzyme being divided into two inactive peptides, LacZa and LacZO. Enzyme activity is restored only when both are present in the same cell, as they spontaneously reassemble into a functional enzyme. This property is exploited in many cloning systems where the presence of the lacZa gene can complement another mutant gene encoding the LacZO. The presence of active ß-galactosidase is detected using X-gal, which produces a characteristic blue colour when cleaved by ß-galactosidase, thereby providing an easy means of distinguishing the presence of cloned product in a plasmid.

 

ß-galactosidase from E. coli has 1024 amino acids and forms a 464 kDa homotetramer.

 

Beta galactosidase assay: the principle

 

A typical beta galactosidase assay includes the following key steps:


  1. Introduction of beta galactosidase expression plasmid into cells by transfection or infection

  2. Incubation for desired time period, then lysis of cells (typically after 24-48h)

  3. Addition of substrate, typically X-gal for colour change reaction

  4. Stop the reaction when adequate colour change has occurred, and read using spectrophotometer at 420 nm.

 

Our product range

 

Oxford Genetics provides a broad range of plasmids containing E. coli beta galactosidase in a variety of configurations, suitable for expression in mammalian cells, bacteria, yeast or insect cells. The structure shown encodes beta galactosidase downstream of the MCS, allowing you to insert your gene into the MCS and create an in-frame fusion protein with beta galactosidase. The 3C 'Prescission' site is also included, in case you wish to remove the reporter after protein production. This vector also contains Puromycin resistance driven by the ubiquitin promoter, for selection in mammalian cells, and kanamycin resistance for selection in bacterial cells. Many diverse plasmid structures are available from our online catalogue (see button below).

 

Our Plasmid Builder facility also provides a simple means for you to design an efficient strategy for any modifications or cloning manipulations to these plasmids. Finally, as always, although we have designed these plasmids for simple and efficient cloning, we are happy to undertake any cloning steps that you prefer to outsource. You can access all these options through the Plasmid Builder’ button below.

 

 

 Colorimetric Reporter Gene Plasmids

 

Alkaline phosphatase SEAP (secreted)

 

Beta galactosidase (cytosolic)

 

CAT Chloramphenicol acetyl transferase (cytosolic)

 

Single
Gene Vectors

 

Chose a promoter to drive the reporter gene

CMV Promoter

 

OG105

OG101

OG594
(+puro)
 

OG104

Minimal CMV Promoter

OG577

OG572

OG576

Other Promoters

 

CAG OG506
(+puro)

 
 

Systems for you to insert your own promoter

 

With polyA upstream of MCS

 

- to minimise background

Prom-MCS-pAa

OG238

OG239

OG240

MinProm-pAb

 

OG248

 

OG249

 

OG250

CMVe-pAc

OG254

OG255

OG256

 

Without upstream pA

 

- better for cloning into retrovirus or lentivirus

Prom MCS (no pA) a

OG371

OG372

OG373

MinProm (no pA) b

OG359

OG360

OG361

CMVe (no pA) c

OG365

OG366

OG367

 

Double
Gene Vectors

 

(place your gene of interest into the MCS, under CMV
promoter control)

 

Chose a promoter to drive the reporter gene

RSV

OG17

OG18

OG19

Ubiquitin

OG157

OG158

OG159

PGK

OG392

OG391

OG393

IRES (Internal Ribosome Entry Site)

FMDV

OG287

   

EMCV

OG297

   

Reporter Fusion Protein Constructs

See separate Table

 

NOTES:

(a)  PromMCS
plasmids have no promoter driving the reporter gene, but they have several
unique restriction sites that can be used to insert the promoter of your
choice.

 

(b)  MinProm plasmids
contain the minimal HSV TK promoter driving the reporter gene, with an
upstream MCS that can be used to introduce specificity-defining sequences
(such as transcription factor binding sites) of your choice

 

(c)  CMVe plasmids
contain the CMV enhancer upstream of the MCS, for your to insert your
promoter of choice. The CMV enhancer should augment its activity.

               

 

View all mammalian
beta galactosidase plasmids