Bio-Rad Ligation and Transformation Module User Manual
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Screening — When bacteria are being transformed with a ligation reaction,
not all of the religated vectors will necessarily contain the DNA fragment
of interest. To produce visible indicators that cells contain an insert,
vectors frequently contain reporter genes, which distinguish them from
cells that do not have inserts. Two common reporter genes are
beta-galactosidase (
b-gal) and green fluorescent protein (GFP)
Some newer plasmid vectors use positive selection, in which the
inserted DNA interrupts a gene that would otherwise be lethal to the
bacteria. If foreign DNA is not successfully inserted into the MCS, the
lethal gene is expressed and transformed cells die. If the foreign DNA
is successfully inserted, the lethal gene is not expressed and the
transformed bacteria survive and divide. Positive selection eliminates
the need for reporter genes, as only cells transformed with vector
containing an insert will survive
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Control mechanism — Most vectors have some control mechanism for
transcription of the antibiotic resistance or other engineered gene. One
of the best-known control mechanisms is the lac operon (an operon is
a group of genes). When lactose (a sugar) is absent in the cell, the lac
repressor protein binds to the lac operon, preventing transcription of the
gene. When lactose is present in the cell, it binds to the lac repressor
protein, causing the repressor protein to detach from the operon. With
the repressor protein no longer bound to the operon, RNA polymerase
can bind and the genes can be transcribed. In this system, lactose acts
as an inducer. (A closely related compound, (IPTG), is often used in the
laboratory as an artificial inducer.) Genes from the lac operon have
been engineered into many cloning vectors
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Size of insert — Plasmid vectors have limitations on the size of inserts
that they can accept, usually less than the size of the vector. Other
vectors have been developed for use if the target DNA is larger, for
example, lambda phage (inserts up to 25 kb), cosmids (inserts up to
45 kb), bacterial artificial chromosomes (BACs; inserts from 100–300 kb),
yeast artificial chromosomes (YACs; inserts from 100–3,000 kb), and
bacteriophage P1 (inserts up to 125 kb)
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