Lab 22 Transformation PDF

Ex. 28 – Transformation Creating Genetic Diversity in Bacteria o Vertical Gene Transfer: sexual reproduction - genetically unique offspring Bacteria can’t do!!! Bacteria are asexual reproduce by binary fission – ge...

Ex. 28 – Transformation Creating Genetic Diversity in Bacteria o Vertical Gene Transfer: sexual reproduction - genetically unique offspring Bacteria can’t do!!! Bacteria are asexual reproduce by binary fission – genetically identical offspring but bacteria can do….. o Horizontal Gene Transfer – transfer of genetic info between adult organisms Transformation – bacteria taking in plasmids from environment Conjugation – bacteria exchanging plasmids Transduction – transfer of DNA via bacteriophages o uptake and/or exchange of antibiotic resistance genes common  bacteria cell bacteria cell Transformation: process by which PLASMIDS are takin in by bacteria Transduction: transfer of DNA Into bacteria using a viral vector Conjugation: process by which PLASMIDS are transferred between bacteria  Ex. 28 – Transformation: creating transgenic organism! transferring “green florescent protein” (GFP) jellyfish protein into e. coli Transformation: uptake of plasmid from environment by bacteria Competent Cells: bacteria that can “transformed” by plasmids o making bacteria “artificially competent”: heat shock salt/heat to “soften” membrane – plasmids go in ice – “stiffen” membrane – plasmids stay in  Ex. 28 – Transformation: creating transgenic organism! transferring “green florescent protein” (GFP) jellyfish protein into e. coli pGLO Plasmid: o ampR resistance gene ampicillin resistance gene o pGLO gene pBAD promotor Arabinose induced promotor  arabinose necessary to activate pGLO gene Plate conditions: o LB o LB +amp (2) o LB +amp / +arab  Ex. 28 – Transformation: Do 2nd (PAIRS) 1. Label tubes “-pGLO” and “+pGLO” 2. Label plates as follows: label LB plate and 1 LB +AMP plate: “-pGLO” label other LB +AMP plate and LB +AMP/+ARA plate “+pGLO” 3. Transfer 250μL of transformation solution into EACH of 2 eppendorf tubes. Place on ICE. 4. Pick single bacterial colony from E. coli plate and swirl loop in the “-pGLO“ tube until bacteria evenly dispersed in solution with no apparent chunks 5. Repeat Step 4 with “+pGLO” tube 6. Using a inoculate loop transfer loop of the pGLO DNA solution (plasmid) into the +pGLO tube. do not add anything to the “-pGLO” tube 7. Incubate the tubes on ice for 10 minutes 8. HEAT SHOCK: place in 42C water bath. Make sure they have good contact with water. Incubate in water bath 50 seconds. 9. Incubate on ice for 2 minutes 10. Add 250μL of LB (luria broth) to both tubes 11. Incubate at room temperature for 10 minutes 12. Pipette 100μL of “-pGLO“ tube onto the LB and LB +Amp plates / spread with “loop” 13. Pipette 100μL of "+pGLO" tube onto the LB +Amp and LB +Amp/+Ara plates – spread with “loop” Green fluorescent protein (GFP) is a natural protein produced by a certain jellyfish species. It glows green under UV light. A bacterial transformation experiment was done using a plasmid containing the gene for GFP. The pGFP experiment attempted to transfer a plasmid containing the gene for the fluorescent green protein (GFP) into E. coli bacteria by transformation. It was done by inoculating a tube containing the pGFP plasmid (+pGFP) and a tube that did not contain the pGFP plasmid (-pGFP) with E. coli bacteria. The “heat shock” procedure was then performed with both tubes. The pGFP gene is spliced into the arabinose gene such that it is only expressed when the arabinose gene is active. Transcription and expression of the GFP gene occurs as part of the arabinose gene transcription. The arabinose gene is an inducible operon that requires the presence of arabinose sugar as the inducer for the arabinose gene. Also included in the plasmid is the gene for ampicillin resistance. Once the procedure was complete the +pGFP tube and –pGFP tubes were plated on the agar plate types below: The –pGLO and +pGLO conditions were plated on different plate types as indicated below. Plate #: Experimental Condition: Plate Type PLATE A -pGLO LB PLATE B -pGLO LB + Amp. PLATE C +pGLO LB + Amp. PLATE D +pGLO LB + Amp. + Arabinose *LB = liquid broth; Amp = Ampicillin; Ara = Arabinose For the plate type described, choose the appropriate option for whether you expect growth / no growth and glowing / not glowing under UV light. A pGFP conjugation experiment was conducted between two species of bacteria: Species A and species B. Bacteria A is the F+ bacteria that contains the plasmid. The plasmid contains genes for ampicillin resistance, arabinose induction of pGFP and pGFP. As in the previous experiment, the pGFP gene is spliced into the arabinose gene such that it is only expressed when the arabinose gene is active. Arabinose sugar is the inducer for the arabinose gene. Bacteria A also contains a chromosomal gene for mercury resistance. Bacteria B is the F- bacteria and has no plasmid prior to conjugation. Bacteria B contains chromosomal genes for streptomycin resistance and vancomycin resistance. Predict whether bacteria B (F- bacteria), AFTER successful conjugation with bacteria A, (F+ bacteria), will grow on the following media types. Will the F- bacteria B grow on the following media? Nutrient agar, +vanc, +strep 1.Growth 2.No growth Nutrient agar, +vanc, +strep, +amp 1.Growth 2.No growth Nutrient agar, +vanc, +strep, +amp, +arab 1.Growth Bacteria A Bacteria B bB bB 2.No growth Nutrient agar, +vanc. +strep, +amp, +merc 1.Growth 2.No growth