Complementation

SpeakerCheng-Lu Shie AdvisorDr. C.Ying Date2008.08.06

Subject
complementation

Principle
Wild type Mutate type

Alpha complementation
Vectors contain a piece of DNA that encodes an alpha fragment of -galactosidase. These vectors exploit a phenomenon called alphacomplementation.

Alpha complementation
The a cell that bears any of a number of deletions of 3 end of the lacZ gene synthesizes an inactive C-terminal fragment of -galactosidase, calld an omega fregment. The a cell that bears a deletions of 5 end of the lacZ encodes an inactive N-terminal fragment of galactosidase, calld an alpha fregment.

Alpha complementation
If a cell contain genes that direct the synthesis of both an alpha and an omega fragment, then, -galactosidase activity is observed. Because the alpha and omega fragments form a complex that has enzymatic activity.

Alpha complementation
Alpha-complementing vectors are used with E.coli strains containing a plasmid F factor that directs the synthesis of an omega peptide. Cells are grown on medium containing IPTG, which inactivates lac repressor and thus derepresses omega peptide synthesis, and X-gal, which is turned blue by the enzymatic activity of -galactosidase.

alpha-complementation

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lac repressor tetramer bound to DNA

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Diauxic growth

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The F Factor
The Ffertilityfactor is a genetic unit found in some strains of E.coli. Possession of F allow a cell to be infected by vectors based on filamentous phages, which bind to proteins in structures called pili elaborated by F-containing cells. Defective lacZ genes that encode the omega fragment of -galactosidase are commonly carried on F factor.

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The F Factor

F Factor conjugation
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The F Factor
The F factor is found in three alternative forms double-stranded circular extrachromosomal plasmid DNAF Plasmid DNA like F bacterial genesF

but also including other

a stretch of linear DNA integrated into various sites on the bacterial chromosomeHfr
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-galactosidase
E. coli -galactosidase is a tetramer of four identical polypeptide subunits, each consisting of 1023 amino acids. This polypeptide is encoded by the first genelac Zof the lac operon.

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-galactosidase
N-terminal

C-terminal -galactosidase structure -galactosidase subunit

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-galactosidase
Whose synthesis is induced by lactose and certain other galactosides, catalyzes two enzymatic reaction Hydrolysis of -D-galactopyranosides on the disaccharide lactose into glucose and galactose. A transgalactosidation reaction in which lactose is converted to allolactose, the true inducer of the lac operon.
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-galactosidase
-galactosidase also interacts with a series of synthetic analogs of lactose in which glucose has been replaced by other moieties. The chromogenic substrates ONPGOD 420 nm and X-gal, and MUG. Inhibitors such as TPEG, which is used for affinity purification of lacZ fusion proteins.

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-galactosidase
-galactosidase is tolerant of deletions and substitutions of amino acid at its amino and carboxyl terminus. Up to 26 amino acids can be removed from the amino terminus and replaced with several hundred or more residues of variety of other proteins without affecting enzymatic activity.

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-galactosidase
The amino and carboxyl domains of the enzyme need not be carried on the same molecule to generate galactosidase activity. Two inactive fragments of the polypeptide chain, one lacking the amino-terminal region and the other the carboxy-terminal region, are able to associate both in vivo and in vitro to form a tetrameric active enzyme.

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Material
PlasmidpBR322, PUC19 BacteriaE.coli C600, E.coli XL1-BlacZ LB with ampicillin plates 100 mM IPTG, membrane filter sterilized 2 X-gal in dimethylformamide or DMSO
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Method
Preparation of plasmid DNA of pBR 322 and PUC19. Preparation of CaCl2-treated competent cells of E.coli XL1-B and C600. Transformation
pUC19 transforms C600 pUC19 transforms XL1-B pBR322 transforms C600 pBR322 transforms XL1-B
pUC19 and pBR322 mixture transforms XL1-B

Negative control
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Method
Before you spread the transformed competent cells on selective medium above, add 10l IPTG and 50l X-gal on the agar surface separately. Transformed competent cells on selective medium. Incubate at 37 for overnight. Observe and explain the results.
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Result

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Result
C600 no lacZ pBR322 no lacZ pUC19 lacZ -fragment pBR322 and pUC19 mixture XL1-B lacZ -fragment white

white

white

complementation blue white and blue

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Thanks for your attention

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