Operon; bacterial promoter; Rbs; DNA polymerase vs RNA polymerase function (lec #1) - Transcription is carried by RNA polymerase that RNA polymerase recognizes to a specific sequence (the promoter) and start making mRNA next to that position. - Usually, typical bacterial promoter carries tow consensus sequences (the sequence that all the organism shares) TTGACA at position of -35. TATAAT at -10. - As mRNA is being transcribed, ribosome can bind to that mRNA and begin translation - Operon: many genes in a bacteria are organized into operons; operon is fuctionally related genes whose mRNA is synthesized in one piece. o Operon makes polycistronic RNA (an mRNA that codes for more than one protein) o Each gene on that mRNA translated separately
- In eukaryotes, DNA -> (txn) precursor mRNA -> splicing introns -> mature mRNA -> proteins Orientation of DNA stands 5’-3’
lux operon structure and genes – bioluminescence, quorum sensing, autoinducer etc
Isolating genomic DNA from bacteria
Functions of specific reagents – go through layers of cell wall - In the diagram I drew….
Cloning strategy – host, DNA source, vector (lec #2)
- Introduce a foreign piece of DNA into another organism - When the host cells replicate that piece of DNA gets copied - Having large number of cells with identical copies of that piece of foreign DNA that DNA can now code for a protein (also possible for the host cells to produce the proteins) - Host: can be bacteria, yeast, mammalian cell, etc
o Bacteria easiest to use: (require simple growth requirements, short generation/ doubling time. (grow rapidly). Easy and cheap to obatain identical cells. Small genome 4.6 million bp for E coli Vector: plasmid, virus, phage, etc (type of vector you use depends on host) DNA source: genomic DNA or cDNA (DNA copied from RNA)