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39 Cards in this Set
- Front
- Back
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Transformation |
The natural method of HGT that allows bacteria to uptake DNA from its environment and expressing those genes contained in that DNA as functional proteins |
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Horizontal Gene Transfer |
Bacteria acquire new DNA from other bacteria of same generation via a process that does not require normal reproductive process |
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Competent |
Cell wall is in a physiological state due to stress to take up DNA fragments |
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pUC18 |
Commonly used plasmid vector that is used for cloning pieces of foreign DNA in bacteria |
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Vertical gene transfer |
Normal reproductive processes involving DNA replication, mitosis or meiosis & fertilization to transfer DNA |
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plasmid |
Circular, extra chromosome DNA molecules that replicate independently of the bacterial chromosome |
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Recombinant plasmid |
Plasmid that carry additional pieces of foreign DNA that have been inserted through genetic engineering |
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Multiple cloning site |
short segment of DNA embedded into LacZ gene of engineered plasmids allowing for the insertion of DNA fragments |
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Plasmid vector |
Naturally occurring plasmid have been specifically modified and developed for lab purposes |
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Vector |
Carries foreign DNA into bacteria cell |
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Antibiotic resistance gene |
Gene that codes for the resistance of antibiotics |
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Bla gene |
Gene which encodes for enzyme pencillinase which is resistant to antibiotic penicillin |
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Penicillinase |
Enzyme that breaks the beta-lactam ring of penicillin |
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Ampicillin |
Antibiotic that is broad spectrum, semi -synthetic penicillin that blocks peptidoglycan synthesis and kills bacteria |
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Beta-galactosidase |
Enzyme that catalyze hydrolysis of lactose & also the artificial substrate X-gal |
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X-gal |
Added to media and colonies that can metabolize the X-gal will produce a blue color |
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LacZ gene |
Gene that encodes for the enzyme Beta-galactosidase, catalyze hydrolysis of lactose & X-gal |
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Calcium chloride |
Chemical solution that stresses a cell to become competent |
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What is the natural purpose of transformation? |
survival of bacterial species so they can quickly acquire new traits in times of environmental stress |
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What three conditions need to be meet for transformation to occur? |
cells need to die, lysis & release DNA fragments into environment; environmental stresses cause other bacteria to become competent; those competent bacteria encounter DNA fragments & take up fragments, integrate them into their own chromosomes by recombination |
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Who developed transformation process in the lab? |
Griffith in 1928 |
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What is the function of lab transformation? |
To move desired DNA molecules in the form of recombinant plasmid into bacterial host to be replicated by transcription & translation into functional proteins |
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What kind of Media is used in lab transformation |
LB broth & LB + Amp + X-gal plates |
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What type of media is LB broth? |
Enriched media for growth of E.coli, selective media for ampicillin to prevent growth of colonies without the penicillinase enzyme, differential media for metabolism of X-gal by Beta-galactosidase enzyme resulting in colonies blue in color |
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In the transformation lab, what bacteria was used? |
competent E. coli |
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What is the lab procedure? |
Mix competent cells & plasmid DNA; Incubate on ice; heat-shock: add LB broth: incubate: plate onto LB plates with and without antibiotic |
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In the transformation lab, once the competent cells & plasmid DNA was combined, it was important to mix? |
allows competent cells and plasmids to intersect |
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Once the cells were mixed, why did we incubate on ice for 30 minutes? |
allows plasmid to bind to cell membranes while preventing cell wall from rupturing while in fragile state |
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Why was the mixutre heat-shocked at 37*C for 5 minutes in a water bath? |
shock to facilitate the uptake of plasmid DNA |
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Why did we add warm LB broth? |
Enriched & complex media for optimum growth of E. coli |
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Why did we incubate tubes for 30 minutes at 37*C in a water bath? |
Optimum temperature of E. coli to facilitate cells to replicate and express the genes on the plasmid |
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What was the result of the LB with E.coli & water? |
Positive control that shows E.coli culture is viable & growing |
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What was the result of LB + Amp + X-gal with E.coli and water? |
Negative control that shows antibiotic is present and inhibiting growth of E. coli. Shows E. coli culture is sensitive to Antibiotic |
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What was the result of LB and E.coli with plasmid? |
Positive control that shows E. coli + plasmid culture is viable and growing |
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What was the result of LB + Amp + X-gal with E.coli and plasmid? |
Shows E. coli cells that are transformed by uptake of plasmid DNA & now have bla gene to make penicillinase enzyme. White colonies represent E. coli colonies with plasmids that have been recombinant |
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What are some important uses of transformation in the lab? |
create specific mutations in a gene, to sequence the DNA, to study gene expression or to make large quantities of proteins |
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How has pUC18 been engineered to be a use in biotechnology? |
Bla gene which encodes for eznyme penicillinase, LacZ gene which encodes for Beta-galactosidase enzyme & Multiple Coloning Sites located within LacZ gene to differentiate successful transformation of recombinant plasmid |
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how can researchers tell the colony contain a recombinant plasmid? |
The colony will be white with recombinant plasmid and blue with non-recombinant plasmid |
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Explain how we could use a plasmid like pUC18 to make bacteria produce a human protein, such as insulin, for use in medicine? |
Cloning gene; copied via PCR with designed primers, DNA fragment inserted into plasmid, uptake to competent E. coli, E. coli take up plasmid via MCS in LacZ gene, E.coli has ability to transcript & translate the protein to product, grow cultures, produce & purify insulin |
