Chapter 11- Biotech | Part 2: Gene Cloning
Hosts
-capable of rapid growth in inexpensive medium -nonpathogenic. -capable of incorporating DNA -genetically stable in culture -equipped with appropriate enzymes to allow replication of vector.
OTHER CLONING VECTORS
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PLASMIDS AS CLONING VECTORS
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SHUTTLE VECTORS AND EXPRESSION VECTORS
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Yeast Artificial Chromosomes (YACs)
200-800 kb of cloned DNA accommodated Replicate like normal yeast
Genome Analysis Vectors
Bacterial Artificial Chromosomes (BACs) Yeast Artificial Chromosomes (YACs)
Insertional Inactivation
LacZ gene is inactivated by insertion of foreign DNA. Xgal doesn't process.. blue color does not develop.
Bacteriophage Lambda as Vector
Modified can be good vectors. Holds MORE DNA than plasmids In vitro can package DNA properly Efficiently infects hosts. USEFUL IN CLONING LARGE DNA FRAGMENTS.
mRNA Translation
Problems: -Bacterial RBS not present in Eukaryotic Genomes. -Two organisms might use codons differently. -Introns of Euk genes not properly expressible in Proks.
Eukaryotic Cell Cloning Vectors
YACs - yeast artificial chromosomes DNA Virus SV40 Adenovirus, Vaccinia virus, baculovirus.
Expression Vectors
allow experimenter to control the expression of cloned genes. -based on transcriptional control -high levels of protein expression possible. -STRONG promoters. -Efficient operators -Effective terminators that prevent other genes on plasmid from being created.
Blue/White Screening
blue - don't have vector with foreign DNA inserted. white - have foreign DNA inserted (both AMP resistant)
T7 Expression Vector
cloned genes get placed under control of T7 promoter. T7 RNA polymerase gene is present under control of easily regulated systems ... ex. Lac Operon T7 Poly recognizes T7 Promoters only. -Host transcription shut down**** -Cloned genes transcribed only.
pUC19
common cloning vector. A modified ColE1 plasmid. -contains amp resistance + LacZ genes. -contains multiple sites within LacZ.
Bacterial Artificial Chromosomes (BACs)
f-plasmid constructed Mutant E.Coli is usually host.
Integrating Vectors
integrate into host chromosomes.
Plasmids
natural vectors with useful properties. -small size, easy to isolate DNA. -INDEPENDENT origin of replication. -multiple copy number. Get multiple copies of cloned gene. -presence of selectable markers.
Cosmids
plasma vectors containing cos site from lambda genome. -can be packaged into lambda virions -LARGE inserts (50 kb) -dont have to transform E.Coli -Phage is more stable than plasmids
Other Plasmid Vectors
some vectors are made for DNA cloning by PCR. some select for recombinant DNA using viability.
Vector Transfer
through chemical transformation or electroporation
Shuttle Vectors
vectors that are stably maintained in two or more host organisms. Bacterial plasmids that get engineered to function in Euks. -EUK origin of replication. -CENTROMERE recognition sequence.