Lecture 24, 11/7 (Dr. Maybruck 9); Bacterial Genetics

Audio for today's lecture is available here.

• • Slide 1 (handout from 11/5) tools and techniques for biotechnology applications: Plate hybridization
• Plate hybridization - Helps identify specific DNA sequence associated with an organism
• You use plate hybridization in order to determine if an organism is present and is harmful by looking at the DNA.
• Take your food sample and streak a plate –-> incubate plate for a day → bacteria grow. From this growth we do gene analysis.
• Place nitrocellulose on top of the colonies (so that they touch) that have grown. Pull the membrane off and it contains smears of the colonies that had grown on the plate. Next you lyse the cells and denature the DNA. Denaturing the DNA causes it to separate into single strands. You then expose the membrane to a gene probe. The gene probe is a sequence of DNA that will complimentary bind to the gene of interest (the one that has the toxins). If there is no harmful gene then the gene probe will not bind. The gene probe has a radioactive label that emits energy. The photographic film picks up the radioactive label (if it is bound to the DNA in one of the colonies) and a mark appears on the photographic film. You can then take the photographic film, lay it over the original colony again, and determine which colonies have the genes of interest.
• • Slide 2 – PCR
• Polymerase chain reaction allows us to amplify specific sequences of DNA.
• Components of a test tube:
• First thing you need is your DNA fragment that you wish to amplify. Then you need DNA polymerase which is responsible for synthesizing the new complimentary strand of DNA (semiconservitive replication). DNA polymerase will not add complimentary bases to make a new strand of DNA without primers. When making a complimentary new strand of DNA you need nucleotides.
• The above components will be put into a thermocycler (instrument that cycles through temperatures).
• 3 steps occurring in one cycle of PCR (the more cycles the more DNA molecules)
• • denaturization – heat can be used to separate the DNA strands to separate at 94ºC
• • priming – temperature is cooled to 50-65ºC. this causes the primers to bind were they need to. This is important because DNA polymerase wont start adding new nucleotides until there is a primer there.
• • Extension – temp is increased to about 70º which causes DNA polymerase to come in and start making new strand of DNA.
• Thermos aquaticus – thermophile. Organism able to survive in extreme heat.
• • Taq polymerase – used often in PCR because its polymerase is used to being at hot temperatures.
• Video explanation titled “Polymerase Chain Reaction” – available on blackboard
• • Slide 3 DNA sequencing
• DNA sequencing: Gives us an idea of what we are dealing with
• Sanger dideoxy method of DNA sequencing
• • Dideoxy nucleotide is missing an oxygen from its 3 carbon sugar.
• In a test tube you have: gene, primers, DNA polymerase, 4 nucleotides (G,C,A,T), 1 modified nucleotide (this is the dideoxy nucleotid missing oxygen from its 3 carbon sugar)
• Once the modified nucleotide is added to the chain then nothing will add on afterwards.
• Video titled “SANGER SEQUENCING” available on blackboard
• • Slide 1 (handout 11/7)
• Recombinant DNA is taking the genetic material of one organism and incorporating it into the DNA of another
• This is a natural occurrence among bacteria. It is called transformation.
• We can use this to our benefit as we can make organisms make proteins, Make transgenic organisms and amplify DNA.

Lecture 23, 11/5 (Maybruck 8); Operons + Bacterial Genetics

You can get the audio for lecture on 11/5 here.

• • Review of last class - check out the animations on blackboard.
  
• • Slide 1 (handout from 10/26) Lactose operon: inducible operon
• Operons (structures found in bacteria (prokaryotes)) – grouping of genes that are adjacent to each other and are involved in coding for one particular phenotype
• Their transcription will be regulated together
• Lac operon (inducible operon) – the only way that transcription is going to occur is if lactose is present.
• Promoter region – where RNA polymerase is binding. Once it binds there it will start transcribing (3’-5’ direction). This is what operons are regulating – with the help of an operater region. This is found “downstream” of the promotor. (downstream indicates that it is in the direction that transcription occurs – 3’-5’ . . . upstream is the opposite.)
• If you want transcription to happen in lac operon you must get rid of repressor protein. The repressor protein has two binding sites one binds to the operating region. Once bound it prevents RNA polymerase from transcribing. The other binding site will bind to lactose. Lactose is the inducer of this operon. Lactose binds to repressor protein and causes it to change protein form. This protein can no longer bind. This allows rna polymerase to begin transcribing. The only time this operon will transcribe is when lactose is present.
• What is being transcribed in this operon?
• • Beta-galactosidase : this enzyme is responsible for breaking down lactose into galactose and glucose.
• • Slide 2 arg operon: repressible operon
• Arg operon – responsible for a group of genes that are responsible for building the amino acid argenine. RNA polymerase comes along and transcribes Arg operon. Argenine acts as a repressor. It associates with a repressor protein (which has two binding sites like the other one). One site is for argenine the other is for the operator region of that operon. When argenine is no longer available to bind to the repressor protein the repressor protein will move away from operater region and allows transcription to occur once more.
• • Slide 1 Application of bacterial genetics (handout11/5/07)
• Biotechnology – manipulating the biochemical processes of an organism to benefit humanity.
• • Example: insulin → there are bacteria that make our human insulin
• • It can also help us identify different bacteria
• • Slide 2 restriction endonucleases
• Restriction endonucleases - Internal cutting of DNA
• The restriction endonucleases are bacteria’s immune system. They try to stifle viruses attempt to inject their DNA into the bacteria.
• They always cut palindrome sequences.
• Sticky ends and blunt edges
• • Sticky ends created by restriction endonucleases. They occur when the DNA is split. These sticky ends go on and bind to other complimentary pairs.
• • Blunt ends are cut clean
• Sticky ends are used in a process called recombinant DNA. Recombinant DNA splices foreign DNA.
• Restriction fragment length polymorphism (RFLP)– a piece of DNA that has been cut by a restriction enzyme. We can use the RFLP to distinguish between different organisms.
• • Slide 3 analysis of DNA + getl electrophoresis
• Polymorphisms – the subtle differences that are occurring between the different nucleotide sequences of the organisms.
• EcorI cuts up certain base pair sequences consistently for different organisms. The cuts leave certain lengths which cause it to be identifiable.
• Gel electrophoresis helps identify cut pieces
• • Add the samples (cut pieces) to gel wells.
• • The gel is exposed to an electrical current which creates a negative charge at one end and a positive charge at the other end. The positive charge is always away from the wells. DNA has an overall negative charge to it. This means that it will want to move away from the negatively charged wells.
• • The fragments then spread out through the gel. Smaller fragments migrate faster. Large fragments move slower.
• • From there you can compare electrophoresis tests to compare and classify different organisms.

Lecture 21, 10/31 (Maybruck 6); Bacterial Genetics

Here is the audio.

• • Slide 1 of handout from 10/29 – bacterial genetics replication, transcription, and translation
• o The study of bacterial heredity discusses passing of traits to subsequent generations and evolution of genetic material (more on slide one of handout from 10/29)
• • Slide 2 levels of structure and terminology
• o Genome – total amount of genetic material in a cell
• • In bacteria and eukaryotes and viruses the genetic material is DNA. In a retrovirus it would be RNA (ex. HIV)
• o Chromosome – includes genes that are critical for the survival of the bacteria/cell.
• • We have diploid chromosome sets (two copies). 23 chromosomes 2 copies . . . 46 total
• • Haploid have only one copy.
• • Chromosome is supercoiled to save space. For it to be copied it will be unwound by a DNA gyrase.
• o Plasmids – another type of genetic material. Plasmids help but are not essential. They allow the bacteria to adapt to a certain situation
• o Gene – a specific sequence of nucleotides. Found within the chromosome they are a specific sequence of nucleotides that will code for a protein.
• o Genotype – genetic makeup of a gene. Genotype is a specific nucleotide sequence of that gene.
• o Phenotype - The protein that was produced by the gene produces a trait and that is called the phenotype
• • Slide 3 DNA
• o Basic unit of DNA is a nucleotide.
• o Nucleotide – includes nitrogenous base which defines the type of nucleotide we have, They have a phosphate group and a sugar group.
• o Hydroxyl group lacking on the ribose sugar of DNA. If you see a hydroxyl group you know you have an RNA.
• o Each separate strand of the DNA (nucleotide) is covalently bound – unequally shared electrons.
• o The phosphate group is covalently bound to an adjacent nucleotide at the #3 carbon.
• o sugar phosphate linkage occurs on outside of helix
• o you ultimately get the double helix
• • slide 4 DNA
• o purines: adenine and guanine
• o pyrmidines: thymine and cytosine
• o two colons two hydrogen bonds 3 colons three hydrogen bonds. A::T G:::C
• • slide 5+6 DNA replication in bacteria: a semiconservative process
• o semiconservative - formation of a new DNA molecule from old DNA strands
• o STEP 1
• • uncoiling the DNA using DNA gyrase
• • separating the DNA molecule into 2 strands – helped by the enzyme helicase (which goes to “A” “T” rich area which is the origin of replication) and single stranded binding proteins (without these the hydrogen bonds would reattach). The A::T rich sight is the origin of replication – it is easier for the helicase to break down the bonds here.
• • The area where the strands are being split is called the replication fork.
• • Slide 7 DNA replication in bacteria: a semiconservative process
• o In order for the synthesis of new nucleotides to be added to the old DNA strand to form a new DNA strand you need RNA primase and DNA polymerase III.
• o DNA polymerase III – before it can add new nucleotides it needs a primer (RNA primase). It works down the strand adding nucleotides according to what it reads on the old strand. It can only read DNA strand in 3’-5’ direction. SO in order for it to replicate 5’-3’ direction it waits for helicase to open up the two DNA strands wide enough so that an RNA primer can get in. The DNA polymerase III uses it to replicate in the opposite direction.
• o RNA primase – adds complimentary RNA nucleotides
• o Processing of the lagging strand creates okazaki fragments.
• o DNA polymerase I (repair polymerase) - removes all of RNA primers and replace it with the correct complementary nucleotides in the newly synthesized strand.
• • Slide 8
• o DNA ligase – connects okazaki fragments
• o Freesciencelectures.com video DNA replication process
  
• 
  
• • Slide 9 transcription and translation overview
• o Gene has a specific sequence of nucleotides that’s going to code for a protein. That DNA within the gene has nucleotides that can be grouped into 3’s (triplets). Each of those groups will code for a specific amino acid. We get a protein that rolls over the DNA and creates an exact copy of the gene in RNA (mRNA). RNA’s grouped together as three is known as a codon (in DNA it is a triplet) which codes for a specific amino acid which is what proteins are made of.
• o DNA → RNA = transcription
• o mRNA → amino acids = translation
• • Slide 10 RNA
• o Look at diagram on the handout.
• o RNA uses uracil (in pyrimidine group) instead of thymine
• • Slide 11 RNA (1 strand of covalently bound nucleotides)
• o mRNA – RNA that is involve in coding for a protein. Provides an exact copy for a gene.
• o tRNA – parts of tRNA that complimentary bind to one another making it look like it has two strands. This creates hairpin structures. On one of the hairpin structures there is a triplet nucleotide sequence. That nucleotide sequence will bind to a specific sequence of mRNA. At the other end of the tRNA a specific amino acid will be attached.
• • tRNA brings the specific amino acid of the mRNA to the codon. It knows because of the anticodon which complimentary binds to the codon.