Microbio 310 1st Edition Lecture 14Outline of Last Lecture I. 11.1 Restriction and Modification EnzymesII. 11.2 Nucleic Acid HybridizationIII. 11.3 Essentials of Molecular CloningIV. 11.4 Molecular Methods for MutagenesisV. 11.5 Gene Fusions and Reporter GenesVI. 11.6 Plasmids as Cloning VectorsOutline of Current Lecture I. 11.7 Hosts for Cloning VectorsII. 11.8 Shuttle Vectors and Expression VectorsIII. 11.9 Bacteriophage Lambda as a Cloning VectorIV. 11.10 Vectors for Genomic Cloning and SequencingV. 12. 1 Introductions to GenomicsVI. 12.2 Sequencing and Annotating GenomesVII. 12.3 Bioinformatic Analyses and Gene DistributionCurrent Lecture11.7 Hosts for Cloning Vectors• Ideal hosts should be: – Capable of rapid growth in inexpensive medium These notes represent a detailed interpretation of the professor’s lecture. GradeBuddy is best used as a supplement to your own notes, not as a substitute.– Nonpathogenic – Capable of incorporating DNA (transformable)– Genetically stable in culture – Equipped with appropriate enzymes to allow replication of the vector • Escherichia coli – Advantages:o Well developed geneticso Many strains availableo Best Known bacterium– Disadvantages:o Potentially pathogenico Periplasm traps proteins• Bacillus subtilis – Advantages:o Easily transformed o Nonpathogenico Naturally Secretes proteinso Endospore formation simplifies culture– Disadvantages:o Genetically unstableo Genetics less developed than in E. coli• Saccharomyces cerevisiae (eukaryote)– Advantages:o Well developed geneticso Nonpathogenico Can process mRNA and proteinso Easy to grow – Disadvantages:o Plasmids unstableo Will not replicate most bacterial plasmids11.8 Shuttle Vectors and Expression Vectors• Shuttle vectors: vectors that are stably maintained in two or more unrelated host organisms (e.g., E. coli and B. subtilis or E. coli and yeast) – Bacterial plasmid engineered to function in eukaryotes• Add a eukaryotic origin of replication• Add a centromere recognition sequence • Expression vectors: allow experimenter to control the expression of cloned genes – Based on transcriptional control – Allow for high levels of protein expression – Strong promoters(allow you to make lots of proteins)• lac, trp, tac, trc, lambda PL – Effective transcription terminators are used to prevent expression of other genes on the plasmid - In T7 expression vectors, cloned genes are placed under control of the T7 promoter- Gene for T7 RNA polymerase present and under control of easily regulated system (e.g., lac) – T7 RNA polymerase recognizes only T7 promoters o Transcribes only cloned geneso Shuts down host transcription • mRNA produced must be efficiently translated and there are problems with this always happening – Bacterial ribosome binding sites are not present in eukaryotic genomes – Differences in codon usage between organisms – Eukaryotic genes containing introns will not be expressed properly in prokaryotes • Vectors exist for cloning in eukaryotic cells – Yeast artificial chromosomes (YACs)– DNA virus SV40– Adenovirus, vaccinia virus, baculovirus• Integrating vectors– Integrate into host chromosome – Stably maintained in cell 11.9 Bacteriophage Lambda as a Cloning Vector • Modified lambda makes a good cloning vector– Well-understood biology – Can hold larger amounts of DNA than most plasmids – DNA can be efficiently packaged in vitro– Can efficiently infect suitable host particles • Replacement vectors are useful in cloning large DNA fragments - Cloning with lambda: 1. Isolating vector DNA from phage particles and cutting it with the appropriate restriction enzyme 2. Connecting the lambda fragments to foreign DNA using DNA ligase 3. Packaging of the DNA by adding cell extracts containing the head and tail proteins 4. Infecting E. coli cells and isolating phage clones by picking plaques 5. Checking the recombinant phage for the presence of foreign DNA 11.10 Vectors for Genomic Cloning and Sequencing • Specialized vectors for genome analysis exist – Bacteriophage M13– Bacterial artificial chromosomes (BACs)– Yeast artificial chromosomes (YACs) • Yeast artificial chromosomes – Can accommodate 200–800 kilobases of cloned DNA– Replicate like normal yeast chromosomes• Human artificial chromosomes (HACs)– Similar to YACs– Segments of human DNA added to BACs– Ex: artificial chromosome for insulin production: nobody has diabetes anymore12.1 Introduction to Genomics - Genome – Entire complement of genetic information – Includes genes, regulatory sequences, and noncoding DNA - Genomics – Discipline of mapping, sequencing, analyzing, and comparing genomes • > 2,000 prokaryotic genomes sequenced or in progress • RNA virus MS2– First genome sequenced in 1976– 3,569 bp (considered small)• Haemophilus influenzae (bacteria) (does not cause the flu)– First cellular genome sequenced in 1995 – 1,830,137 bp 12.2 Sequencing and Annotating Genomes• Sequencing: determining the precise order of nucleotides in a DNA or RNA molecule • Sanger dideoxy method – Invented by Nobel Prize winner Fred Sanger – Dideoxy analogs of dNTPs used in conjunction with dNTPs– Analog prevents further extension of DNA chain (has H on 3’-carbon of base instead of OH)– Bases are labeled with radioactivity; use radioactive DNA primer– Gel electrophoresis is then used to separate reaction products • Large-scale sequencing projects have led to automated DNA sequencing systems– Based on Sanger method– Radioactivity replaced by fluorescent dye (fluorescent sequencing)• 454 sequencing system – Recent technological advance – Generates data 100x faster than Sanger method – 454 relies on two major advances• Massively parallel liquid handling and pyrosequencing – Light is released each time a base is added to DNA strand – Instrument actually measures release of light – Can only handle short stretches of DNA • Virtually all genomic sequencing projects use shotgun sequencing (order and orientation of DNA fragments are unknown)– Entire genome is cloned and resultant clones are sequenced – Much of the sequencing is redundant (want this to make sure you get absolutely everything)– Generally 7- to 10-fold coverage • Computer algorithms used to look for replicate sequences and assemble them - Occasionally assembly is not possible - Closure can be pursued using PCR to