Molecular Biology II Fall 2024 PDF

Molecular Biology II 1. Cloning 2. Protein expression and purification 3. DNA sequencing 4. CRISPR/Cas gene editing 1  Methods in Molecular Biology I 1. Recombination and gene deletion 2. Using bacteria to assay mutagens 3. Mutant libraries and transposon...

Molecular Biology II 1. Cloning 2. Protein expression and purification 3. DNA sequencing 4. CRISPR/Cas gene editing 1  Methods in Molecular Biology I 1. Recombination and gene deletion 2. Using bacteria to assay mutagens 3. Mutant libraries and transposon mutagenesis 4. PCR, qPCR, RT-qPCR 5. Gel Electrophoresis 6. Hybridization of Nucleic Acids a. Northern hybridization b. In situ hybridization 2 Gel electrophoresis Agarose gel shown here Takes advantage of the inherent charge of nucleic acids Linear molecules fractionate through the agarose matrix based on size 3  Gel electrophoresis What would happen If we switched the electrodes? https://www.yourgenome.org/facts/what-is-gel-electrophoresis image credit Genome Research limited accessed April 9 2019 4  Use a size standard, lane A, to figure out the sizes of other fragments Note the alignment of the gel in the electrical field 5 http://info.gbiosciences.com Visualize DNA using DNA dye and 6 Nucleic Acid Hybridization Relies on the ability of complementary (or nearly complementary) strands of NA to hydrogen bond Find a clone that contains a gene of interest Identify related DNA sequences in other genomes See if a gene is expressed and how much it’s expressed Identify specific RNA or DNA sequence in intact cells! 7  Northern Hybridization Here RNA samples are probed with a gene probe 8 In situ Hybridization Use a DNA probe to find a cell that contains that sequence (or location of that sequence within a cell) 9  Molecular Biology II 1. Cloning 2. Protein expression and purification 3. DNA sequencing 4. CRISPR/Cas gene editing 10  How can we purify large quantities of a human protein from bacteria? Human insulin, licensed by the FDA in October 1982, was the first recombinant pharmaceutical approved for use in the United States. 11  Naturally occurring plasmids Harbor non-essential genes (but these genes may enhance survival) Size range: ~1kb to >1 Mb Single-copy or hundreds of copies in a cell Replicate and Segregate into offspring cells independent of chromosome Important tools in biotechnology – Easy to isolate away from chromosomal DNA – Easy to get back into E. coli 12  Restriction Enzymes Restriction Endonucleases – Endo = “within” Endonucleases break the sugar-phosphate backbone – Exo = “outside” Exonucleases digest starting at the free end of a nucleic acid molecule, removing each base by breaking the sugar-phosphate backbone (e.g. proofreading activity of DNA polymerases) Restriction endonucleases are produced in diverse bacteria and archaea Cuts only double-strand DNA targets, will not cut ssDNA, single-strand RNA or double-strand RNA First discovered because they “restricted” growth of phage in bacterial hosts - part of a system to protect a cell from incoming DNA 13  EcoRI “eco R one” “Eco”= E. coli “R” = strain “I” = first enzyme from this strain Leaves “sticky ends” Estimated cut frequency in genomic DNA: one site per 46 or per 4096 base pairs In real" DNA, depends on GC content etc. 14 15 DNA methylation affects RE activity 16  e.g. Dam methylase (recognition: GATC) CH3 ATGTCTAGATC ATGTCTAGATC CATAGATCTAG CATAGATCTAG CH3 XbaI XbaI CH3 ATGT CTAGATC ATGTCTAGATC CATAGATC TAG CATAGATCTAG C32 Xba1 from Xanthomonas badrii 17 Cloning into a Plasmid Vector 18  Compatible ends of DNA fragments Cut foreign DNA and plasmid with the same restriction endonuclease(s) to make sticky ends that will link up (compatible) Ligation: usually using a phage DNA ligase, and some source of energy (ATP) to drive the reaction, heals the plasmid DNA sugar-phosphate backbone ps://www.khanacademy.org/science/biology/biotech-dna-technology/dna-cloning-tutorial/a/restriction-enzymes-dna-ligas 19 The restriction site SmaI has the blunt end sequence: CCC GGG Would cloning into a plasmid be more efficient using blunt ends or sticky ends? 20 21 Essential components of a plasmid for cloning and gene expression 1. Origin of replication 2. MCS 3. promoter 4. Selectable marker 22  MCS- region with many RE sites 23 (also controls plasmid copy number) The lac promoter is a common promoter for gene expression LacZ: b- galactosidase LacY: lactose permease LacA: thiogalactoside transacetylase LacI: repressor 24 The lac promoter is a common promoter for gene expression IPTG IPTG: gratuitous inducer of the lac operon Similar to lactose, but not metabolized by the cell 25  Blue-white screening lacZ Beta-galactosidase 5,5'-dibromo-4,4'-dichloro-indigo X-gal LacZ encodes the enzyme beta-galactosidase Beta-galactosidase breaks down x-gal into a chromogenic molecule 26  Blue-white screening lacZ Beta-galactosidase X-gal 5,5'-dibromo-4,4'-dichloro-indigo Blue colonies Gene x X X lacZ Beta-galactosidase X-gal 5,5'-dibromo-4,4'-dichloro-indigo 27 white colonies  MCS placed in lacZ so that if insert goes into the plasmid the colonies will not have functional lacZ 28  Blue-white screening Transform E. coli with ligation products Plate on good growth medium with ampicillin, IPTG and X-gal Blue colonies – LacZ has been made – lacZ gene has been reconstituted White colonies – functional LacZ is not made – a chunk of DNA has been inserted into the plasmid and disrupted lacZ https://www.thermofisher.com/us/en/home/life-science/cloning/cloning-l earning-center/invitrogen-school-of-molecular-biology/molecular-cloning /cloning/traditional-cloning-basics.html 29 What would be the most probable answer if you had no white colonies on your plate? A. The X–gal is not being expressed properly B. You forgot to add the ampicillin C. Your plasmid has no inserts D. You added too much IPTG! 30 31 Antibiotic selection of cells with plasmid 32  17 Molecular Biology II 1. Cloning 2. Protein expression and purification 3. DNA sequencing 4. CRISPR/Cas gene editing 33 Expression vectors for E. coli Strong promoter (T7 phage gene promoter) to drive expression of your cloned gene – Need to use this in an E. coli strain engineered to express the T7 RNA polymerase Strong terminator so only your gene is overexpressed RBS so protein is made efficiently His-tag shown here – to aid in protein purification 34  Expression vectors for E. coli Induce lac T7 RNA polymerase Gene promoter with product IPTG Gene for lac T7 RNA lac operator polymerase promoter T7 promoter Cloned gene pET plasmid Chromosome lacl 35 Protein purification 36  Protein purification His tag binds to the Nickel resin All non-His-tagged proteins wash through, while the tagged protein remains bound 37  17 Molecular Biology II 1. Cloning 2. Protein expression and purification 3. DNA sequencing 4. CRISPR/Cas gene editing 38 Sequencing- Sanger method 39 Sequencing- Sanger method 40 Sequencing- Sanger method 41 ** https://www.youtube.com/watch?v=HMyCqWhwB8E Next generation sequencing 42 For a few short For sequenci millions ng reads of sequenci ng reads! 43 Meta-genomics 44 Match the sequencing tools we have discussed to the following situations: Sanger sequencing All genes from a microbial community living in Next Generation a coastal marine Sequencing environment To look at one Meta-genomics specific gene To search for an outbreak of a microbial pathogen in a hospital 45 46  17 Molecular Biology II 1. Cloning 2. Protein expression and purification 3. DNA sequencing 4. CRISPR/Cas gene editing 47  CRISPR/Cas Clustered Regularly Interspaced Short Palandromic Repeats “bacterial adaptive immunity” 48  CRISPR/Cas Clustered Regularly Interspaced Short Palandromic Repeats “bacterial adaptive immunity” spacer spacer pacers contain DNA from phage that have infected the bacterium in the past 49  CRISPR/Cas Clustered Regularly Interspaced Short Palandromic Repeats “bacterial adaptive immunity” spacer spacer acers contain DNA from phage that have infected the bacterium in the past Cas9 endonuclease 50 CRISPR/Cas spacer spacer 51 CRISPR/Cas spacer spacer 52 CRISPR/Cas spacer spacer 53 CRISPR/Cas X spacer spacer 54 CRISPR/Cas 55  CRISPR/Cas gene editing Repair template Can contain NEW desired sequence 56 Sickle cell anemia is caused by a point mutation in the gene for hemoglobin (glu -> val) Cells from the patient were edited so that they would turn on expression of fetal hemoglobin The fetal hemoglobin compensates for the mutated hemoglobin 57 58  Summary 1. Cloning – Restriction enzymes are used to digest a plasmid and clone genes into the plasmid – Plasmids have 4 major features 1. Origin of replication 2. Selectable marker 3. Promoter to drive expression of the gene 4. Multi cloning site next to the promoter 2. Protein expression and purification – Used to study the activity of a protein in vitro – Easiest way to purify a protein is to put an affinity tag on it (such as a His-tag) 3. DNA sequencing – Sanger sequencing – Next-gen sequencing 4. CRISPR/Cas – Bacterial defense system against phage – Has been used to edit the human genome 59