Difference between revisions of "Part:BBa K1444017"
 
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<partinfo>BBa_K1444017 short</partinfo>
 
<partinfo>BBa_K1444017 short</partinfo>
  
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Beta-galactosidase (EC 3.2.1.23) is a classic enzyme most commonly used in blue-white colony screening when used in specialized vectors. This part consists of the full length beta-galactosidase sequence which can be inserted downstream of a promoter to produce the tetrameric, 47.4kDa enzyme.
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Beta-galactosidase (EC 3.2.1.23), or LacZ, is a classic enzyme most commonly used in blue-white colony screening when used in specialized vectors. This part consists of the full length beta-galactosidase sequence which can be inserted downstream of a promoter to produce the tetrameric, 47.4kDa enzyme. This part was amplified from a well-used expression vector from a molecular biology lab, and cloned into the BioBrick vector <partinfo>pSB1C3</partinfo>.
  
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Beta-galactosidase cleaves the beta-glycosidic bond of a galactose bound to an organic moiety; the physiological function is to break down beta-galactosides, such as lactose, into it's respective monnosaccharides. Beta-galactosidase can act as a reporter when provided substrates such as X-gal. The cleavage of X-gal releases galactose and the chromophoric, subtsituted indole moiety. The most common form of X-gal is 5-Bromo-3-indolyl &#946;-D-galactopyranoside, which produces an intense blue pigment. Other versions of X-gal may be used to produce other color pigments.
 
  
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Typically 40-60uL of a 20ug/mL X-gal solution, dissolved in dimethyl sulfoxide or dimethyl formamide, is spread on a plate and allowed to dry before plating the bacteria expressing beta-galactosidase. X-gal may also be added to liquid cultures or incorporated into solid agar media.
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K1444017 SequenceAndFeatures</partinfo>
  
  
 
===Usage and Biology===
 
===Usage and Biology===
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This part was amplified from a commonly used expression vector from a molecular biology lab, and cloned into the BioBrick vector <partinfo>pSB1C3</partinfo>. With expression via an appropriate promoter, this part catalyzes the cleavage of lactose into glucose and galactose. In the lab, it most commonly used to cleave the lactose analog [http://en.wikipedia.org/wiki/X-gal X-gal], which after cleavage produces a deep blue colour.
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[[File:Calgary_lacZ_fig.png|500px|thumb|right|'''Figure 1:''' Examples of the colour produced by this protein in ''E. coli'' on solid media.]]
  
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40 uL 20 mg/mL X-gal (2%)
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40 uL of 1mM IPTG dissolved in DMSO.
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Beta-galactosidase cleaves the beta-glycosidic bond of a galactose bound to an organic moiety; the physiological function is to break down beta-galactosides, such as lactose, into it's respective monnosaccharides. Beta-galactosidase can act as a reporter when provided substrates such as [http://en.wikipedia.org/wiki/X-gal X-gal]. The cleavage of X-gal releases galactose and the chromophoric, substituted indole moiety. The most common form of X-gal is 5-Bromo-3-indolyl &#946;-D-galactopyranoside, which produces an intense blue pigment. Other versions of X-gal may be used to produce other color pigments.  
  
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Typically 40-60uL of a 20ug/mL X-gal solution, dissolved in dimethyl sulfoxide or dimethyl formamide, is spread on a plate and allowed to dry before plating the bacteria expressing beta-galactosidase. X-gal may also be added to liquid cultures or incorporated into solid agar media. These images illustrate the colour that this protein is able to produce in ''E. coli'' on standard LB plates.
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</p>
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K1444017 SequenceAndFeatures</partinfo>
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Latest revision as of 03:38, 18 October 2014

Full length beta-galactosidase (LacZ)

Beta-galactosidase (EC 3.2.1.23), or LacZ, is a classic enzyme most commonly used in blue-white colony screening when used in specialized vectors. This part consists of the full length beta-galactosidase sequence which can be inserted downstream of a promoter to produce the tetrameric, 47.4kDa enzyme. This part was amplified from a well-used expression vector from a molecular biology lab, and cloned into the BioBrick vector pSB1C3.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Usage and Biology

Figure 1: Examples of the colour produced by this protein in E. coli on solid media.

Beta-galactosidase cleaves the beta-glycosidic bond of a galactose bound to an organic moiety; the physiological function is to break down beta-galactosides, such as lactose, into it's respective monnosaccharides. Beta-galactosidase can act as a reporter when provided substrates such as [http://en.wikipedia.org/wiki/X-gal X-gal]. The cleavage of X-gal releases galactose and the chromophoric, substituted indole moiety. The most common form of X-gal is 5-Bromo-3-indolyl β-D-galactopyranoside, which produces an intense blue pigment. Other versions of X-gal may be used to produce other color pigments. Typically 40-60uL of a 20ug/mL X-gal solution, dissolved in dimethyl sulfoxide or dimethyl formamide, is spread on a plate and allowed to dry before plating the bacteria expressing beta-galactosidase. X-gal may also be added to liquid cultures or incorporated into solid agar media. These images illustrate the colour that this protein is able to produce in E. coli on standard LB plates.