BIO 326R 1st EditionExam # 3 Study Guide Lectures: 16 -22 Lecture 16 (October 9) Making PlasmidsSteps to Putting a Plasmid into E. coli- conjugationo plasmid types: self transmissible and mobilizable plasmid (know what this entails)o know function of pili know function of cytoplasmic bridgeo genes used: tra= produce pilus, trans-acting and mob= transfer plasmid for replication in transconjugate and recirculizes daughter cell then replicated to produce dsDNA, protein relaxase (know function), OriT (know function)- transformationo natural (competent)—not good for plasmids gram+ and gram-o artificial (chemically competent)o electroporation plasmid and E. coli between electrical field electrical field transiently opens holes in E. coli and plasmid entersLecture 17 (October 11) Transduction and TransposonsSteps to putting a plasmid into E. coli continued- transductiono generalized transduction bacteriophage= virus (know definition/function) lytic bacteriophage= lyses prokaryotic bacteria- adsorbs to succeptible bacteriumo phage genome enters bacterium, genome directs bacterium’s metabolic machinery for phage components- phage head or caspid occasionally assembles around fragment of donot or around a plasmid instead of a phage genome by mistake- bacteriophages are released- phage carrying donor DNA adsorbs to recipient- phage inserts donor DNA into recipient- donor DNA is exchanged for some of recipient’s DNA- understand homologous recombinationTransposons- = jumping genes- Used as a screening tool—understand how with these components:o IR, sticky ends, transposase (trans acting)Lecture 18 (October 14) TransposonsTransposons continued- know the properties of a good transposon and why they are importanto random insertion, antibiotic resistance, transpose at high frequency, self transmissible, suicide plasmid- drawback: can’t study nonessential genesTranscriptional Regulation- how bacteria regulate behavior- targeted mutagenesis—understand the processo make plasmid with PCR, homologous recombination antibiotic resistant gene- regulation factors—know function of:o sigma70, RpoS, transcription factors (activators and repressors)o operator= sequence that transcription factors bind toPromoter Activity (more on this next lecture)- reporter geneso tells you about transcription—can use lacZ (encodes beta-galactosidase) and observe regulation- translational functiono SD comes from gene of interest to tell you about transcription and translation, because tells you about the promoter and SDLecture 19 (October 16) Reporter GenesReporter Genes- beta galactosidase encoded by lacZo know functiono ONPG, GFP, luxCDABE Know how they are used to test/visualize different things Know advantages and disadvantagesTriparental and Biparental Conjugation- Methods of introducing a plasmid into an acceptor- three strain vs. two strain matingo understand the process of eachLecture 20 (October 21) PhylogenyPhylogeny- =relatedness/ morphological characteristicso Gram stain, cell shape, color, colonies, biochemical tests- Diversity in bacterial species each individual in a bacterial species varies greatly from another individual within that same species= strainsCarl Woese- know what semantide means (sens- containing units)- know how they utilized 16s rRNA sequencing- came up with the 3 domains of life- 16s rDNA sequencing is easier than rRNA, highly conserved, but differs at variable regionso Use variable regions to determine diversity within phylogeny and used in metageneticsMetagenetics- =analysis of the genetic material from a population of microbes, tells you what bacteria are where- Know definition of microbiota and microbiome- Gut microbiota is commonly tested because:o Stable environment, easy to sample, large microbial population, good model animals (mice gnotobiotic)- Know obesity experiment with mice and idea behind it- Know the effect of antibiotics on the metabolic capacity of microbiota- Know how microbiota cures work (fecal transplant)Lecture 21 (October 23) SequencingSequencing- Sanger and Gilbert—Sanger sequencingo Know how this workso Know how they used ddNTPs ddNTPs terminate synthesis when incorporated- random sequencing approacho Stack together to align sequence and eventually obtain a whole genome from thearrangement of random sequence fragments- Whole genome shot gun sequencingo Take apart the whole genome at once, determine sequence of the fragments, putthe entire genome back together- Hierarchical shot gun sequencing o Take apart pieces of the genome, put together the fragments of the pieces, put the pieces together, until the entire genome is in order- Know the various species that were sequenced first - Illumina—a process for sequencing that allows large amounts of sequence to be read very efficiently- Know benefits of having the genomeo i.e. decreased cost, sequencing genomes of individuals (benefits medicine), facilitates comparative genomics, makes sequencing easy in microbiologyLecture 22 (October 25) Quorum SensingQuorum Sensing- =bacterial cell process that counts and determines cell density in order to regulate or perform a function- Know the symbiotic relationship between Vibrio fisheri and squido Bioluminescenceo Counter illumination- Functions of luxI and luxRo luxI= homoserine lacto, makes the signalo luxR= transcriptional regulator that binds to homoserine lacto to turn on luminescence- toxic bacteria use quorum sensing as well, but use it to control toxin levels instead of luminescence- antivirulent strategies of bacterial communities—biofilmso biofilms= bacteria attach and coagulate to a surfaceo highly antibiotic resistanto also use quarum