lay Davis, 8/11/12 Molecular Biology Recombinant DN technology (So... you want to be a genetic engineer) Introduction, or, games to play with DN You have learned something of the structure of DN, its replication, and how the information is read by the cell to produce a functional molecule, often a protein. In this module we use the fluorescent green protein (F) that is made in a jellyfish as a starting point for exploring the techniques and tools of genetic engineering. The basic challenge is to transfer the genetic information from the jellyfish to bacteria such that the bacteria are fluorescent. In doing so we will pick up the basic principles and tools of genetic engineering and review the basic molecular biology of prokaryotes and eukaryotes. Reading Lehninger, 4th ed, pgs 306-322 The ell, 3rd ed., pgs 104-122 Need to know and understand: rocesses DN purification layers phenol extraction Transformation chemical (al 2 ) electroporation loning Recombinant DN enomic DN library Expression library Vectors - These are specialized pieces of DN that can carry, replicate and transfer pieces of DN that we insert in their respective host organisms. Typically in bacterial hosts: lasmid, osmid, hage λ, Bacterial rtificial hromosomes (Bs). lasmids, cosmids and BS all contain an ori, antibiotic resistance gene and a cloning site (MS). Restriction endonucleases T4 DN ligase 1
cdn DN Sequencing Reverse transcriptase E. coli DN ligase RNase H DN polymerase dnts rimer Template dnts ddnts DN polymerase el electrophoresis: Forward force on DN depends on charge of DN charge on DN depends on length mass of DN depends on length frictional force on DN depends on length of DN, and on conformation of DN, and on concentration of matrix log 10 (size) = -K 1( d) K 2 where: size = size of DN fragment in base pairs or kilobasebpairs d = migration distance (mobility) from start (origin) olymerase hain Reaction (R) rimer Template dnts thermostable DN polymerase thermal cycler olony hybridization (screening) preparing probe nitrocellulose replica denaturing hybridizing Hybridization Kinetics (This is the basis for southern and Northern blots and in situ hybridization) 100 o T( o ) <T m ssdn ssdn -> dsdn t The toolbox Enzymes - The enzymes and their activities listed on the following pages are our basic tools of molecular biology. In general each tool will perform a specific action on the target molecule, often DN or RN. Refer to this list as we go through procedures, collect and remember the activities as we go though the lessons. You do not need to remember them all, just the ones we use. 2
Enzyme activities: 1. ->3' polymerase activity T α γ β α γ β 2. 3' -> exonuclease activity T T 3.endonuclease activity 4. ->3' exonuclease activity 5. phosphatase activity 3
6. kinase activity γ β α γ γ β α 7. Ligase activity 8. ->3' RN polymerase activity U U 4
Enzymes E. coli DN polymerase I holoenzyme (ol I) activity: -3' DN polymerase substrate: primed DN activity:3'-> exonuclease substrate: dsdn, ssdn note: slower than polymerase activity activity: -3' exonuclease substrate: dsdn, ssdn note: very slow reaction on ds DN T4 DN polymerase or Klenow fragment of ol I activity: -3' DN polymerase substrate: primed DN S1 nuclease activity: endonuclease template: ssdn or nicked dsdn note: very active, since dsdn temporarily loses some base pairing due to thermal energy, S1 nuclease will eventually degrade dsdn. RNse activity: endonuclease substrate: ssrn RNase H activity: endonuclease, exonuclease substrate: dsrn or RN of DN/RN hybrid activity:3'-> exonuclease substrate: dsdn, ssdn T4 DN ligase: activity: Ligase substrate: dsdn with blunt or cohesive ends or nicked dsdn. E. coli ligase activity: Ligase substrate: Double stranded DN with cohesive ends or nicked dsdn Reverse transcriptase activity: -3' DN polymerase template: primed DN or RN DNase I activity: endonuclease substrate:dsdn or ssdn T7 or T3 or S6 polymerases activity: ->3' RN polymerase substrate: dsdn with T7 or T3 or S6 promoter sequence Restriction endonucleases activity: double stranded endonuclease substrate: dsdn with recognition sequence...nntnn... eg:...nntnn... BamHI T4 polynucleotide kinase activity: kinase template: of DN or RN...NN...NNT NN... TNN... Bacterial lkaline phosphatase (B) alf-intestinal phosphatase (I) activity: phosphatase template: phosphate of DN or RN (ss or ds). 5
The Story of a rotein, its Travels, and How it hanged on the Way. The reen Fluorescent rotein (F) is made by a jellyfish. Two views of the structure of F as determined by X-ray crystallography. The gold arrows are b-sheets. 6
The information to make F can be moved to other organisms. E. coli Fluorescent E. coli colonies on a petri dish Fluorescent E. coli under the microscope If you play with the sequence of the F protein, you can change is color. 7
The F sequence can be added to other eukaryotic cells and animals 8