BIO 326R 1st Edition Lecture 16 Outline of Last Lecture I. Plasmid compatibilitya. Incompatibility groupsb. Partitioning systemII. Cloning into a plasmida. PCROutline of Current Lecture I. Making a Plasmida. Conjugation, transformation, transductionII. Conjugationa. Self transmissibleb. Mobilizable plasmidc. Genes usedi. Tra genesii. Mob genesiii. OriTIII. Transformationa. Natural, artificial, electroporationCurrent LecturePutting a plasmid into E. coli- Conjugation, transformation (most common), transduction (least used)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.Conjugation- Transconjugate= bacteria that receives DNA via conjugation- 2 types of plasmids usedo Self transmissible= contains all the function necessary for conjugationo Mobilizable plasmid= contain some but not all functions necessary for conjugation- Pili= protein protrusions used to attach a donor to a recipient to pull bacteria close together forms a cytoplasmic bridgeo Cytoplasmic bridge= DNA is duplicated transferred via bridge between bacteria- Genes usedo Tra genes= responsible for producing the pilus (~10 nm wide) Pilus attaches to neighboring cells, neighboring cells retract, brings cells close together for gene transfer trans acting - diffusible, don’t need to be on plasmid being transferredo mob (mobilization) genes= prepare plasmid for transfer, ultimately allow plasmid to be replicated in the transconjugate protein relaxase= encoded within mob genes, critical for rolling circle replication- nicks the DNA and covalently binds to the 5’ end of rolling circleo OriT site= site of nicking, origin of transfer, cis acting Self transmissible=> OriT, mob+, tra+ Non self transmissible (mobilizable)=> OriT- Recircularizes the daughter cell then utilizes an RNA primer on thesingle stranded DNAo Primase encoded by the plasmid sent from the mother to the daughter cell- then replicates to produce a double stranded DNATransformation- natural transformation (competence)o take up DNA from the environment gram+= streptococcus pneumoniae binds dsDNA outside the cell and brings ssDNA into the cell gram-= hemophilus influenzae binds DNA with specific sequence (5’-AAGTGCGGTCA-3’)-- can bring in dsDNA, DNA receptors have sequence specificity NOT GOOD FOR PLASMIDS- artificial transformation (chemically competent)- electroporation (plasmid and E. coli between metal plates; electrical field)o electrical field transiently opens holes in E. coli, plasmid
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