MCDB 1041 Class 27. Making recombinant DNA and using it

Transcription

1 MCDB 1041 Class 27 Making recombinant DNA and using it Learning Goals Explain why and how bacteria can be easily used to make copies of human DNA. Compare the two methods for making lots of copies of DNA: PCR and bacterial amplification. Given information about a bacterial plasmid and a piece of DNA, propose how you would cut and combine the two to create recombinant DNA Quiz Friday Review session: Thursday at 12:30 will location

2 PCR is good for making lots of copies of a specific region of DNA However, it is most effective for smallish pieces of DNA (up to 10 Kb) When scientists want to make copies of large pieces of DNA, they often employ a different technique The advantage of the next technique is that pieces of DNA can be isolated, manipulated, and then amplified 18-2

3 Making recombinant DNA Restriction enzymes cut DNA at particular locations Scientists can put different pieces of DNA together (by cutting with restriction enzymes and then sealing the pieces back together) 18-3

4 Why make recombinant DNA? Study how a gene (or its protein) normally work delete genes express a new protein in a chosen location ( Mr. Green Genes at right!) introduce specific mutations into a gene Produce a lot of a specific protein medical uses (insulin, human growth hormone) Produce an altered protein not normally produced by an organism genetically modified crops gene therapy 18-4

5 Putting two pieces of DNA together requires that they have complementary sequences that can pair Isolate gene of interest by restriction enzyme digest (orange piece of DNA) Cut another piece of DNA with the same restriction enzymes (grey piece) The two pieces of DNA have complimentary sticky ends Add the enzyme ligase, and the two pieces of DNA become one piece 18-5

6 This circular piece of DNA is called a plasmid (more in a minute) They exist naturally in bacteria They get replicated just like a regular chromosome within the bacterium 18-6

7 Restriction enzymes and DNA (review) This DNA sequence can be cut by 4 differentrestriction enzymes E=Eco RI P=Pst1 B=BglII E P B H B H=HindIII Each line line represent demarcates 1 Kilobase (1KB). 1 Kilobase You can (1Kb) amplify this 12 KB seq The restriction enzyme sites are shown in detail below If you cut two piece of DNA, one with E and one with H, what can you do next? a. Paste the two pieces together b. Paste only E cut pieces or H cut pieces together c. Nothing once cut, DNA cannot be rejoined G* A A T T C Eco RI restriction site C T T A A *G C T G C A*G G *A C G T C 18-7 Hind III restriction site

8 E P B H B Each line demarcates 1 Kilobase (1Kb) Each line represent 1 Kilobase (1KB). You can amplify this 12 KB seq If this sequence of DNA were cut with both E and H restriction enzymes, how many pieces would be created? a. 1 b. 2 c. 3 d

9 E P B H B Each line demarcates 1 Kilobase (1Kb) In the gel below, if the gene of interest is in between the E and H restriction enzyme sites, which band on the gel represents that piece? H+E Ladder(DNA of known sizes for referen ce) Each line represent 1 Kilobase (1KB). You can amplify this 12 KB seq 12 KB 11 KB 10 KB 9 KB A B C 6 KB 3 KB 1 KB 18-9

10 Features of Bacterial Plasmids Bacterial plasmids are circular, have a site to initiate replication, and can serve as a carrier for another piece of DNA Can be introduced back into bacteria, which will make many copies of the plasmid Essential Features Series of unique restriction sites Selectable Marker AmpR ampicillin resistance gene Bacteria with AmpR will not die in the presence of ampicillin, while other bacteria will ORI origin of replication (for bacteria) allows the plasmid to be replicated in bacteria 18-10

11 Bacterial cells have to incorporate the plasmid Transformation AmpR Plasmid+ gene + Competent bacteria Chemically treated so they will take up DNA Few cells are transformed Transformation is a rare event, so you need a way to kill off all the cells that aren t transformed, leaving only the cells that contain your gene.

12 Select for transformed cells the plasmid contains an ampicilin resistance gene AmpR when a plasmid has this gene, it can make a protein that breaks down ampicilin, preventing this antibiotic from killing the bacteria Plate the bacteria on media that contains ampicilin If you grow bacteria on a plate that has ampicillin, which bacteria will survive? a. All bacteria b. Bacteria that contain the plasmid c. No bacteria media + ampicilin plasmid media + ampicilin 18-12

13 Finally, break open these bacterial cells and remove the DNA from the plasmid Or, allow the bacterial cells to make protein from the DNA, then isolate the protein, which can be used to treat people who can t make the protein This technique is used to make insulin for diabetics 18-13

14 E P B H B Psy gene: Coding from E to H Each line represent 1 Kilobase (1KB). You can amplify this 12 KB seq E P B H B B P B E Crt gene. Coding from P to E. If you want these two genes to be hooked together in sequence, crt, then psy, what can you do? a. Mix the DNA all together, cut with E, P and H. Separate on gel, ligate pieces b. Keep DNA separate. Cut with E and H for one, E and P for other. Separate on gel. Ligate. c. Run gel to separate DNA. Cut with E and H for one, E and P for other. Ligate d. Run gel to separate DNA. Mix all together, cut with E,P,H, then ligate

15 E P B H B Eco RI 9:/$..$ %&'()&'*+,,'(-$ Each Psy line gene: represent Coding 1 Kilobase from (1KB). E to You H can amplify this 12 KB seq.('*-"+(-$ (-/-01+2/-$*+"3-"$ Amp R 4#,5$...$ B P 60'$7.$ E P B H B E B %(1$8$ 9:/$..$ Crt gene. Coding from P to E.!"#$ Once you ve joined your genes of interest together, you need to get them into this plasmid. How should you cut the plasmid so it can incorporate the DNA and be grown in bacteria with ampicillin in the environment? a. With P and E b. With H and E c. With B d. With P and H 18-15

16 Practice: Here is a bacterial plasmid and a piece of human DNA that contains a gene called tol. You want to join the tol gene and the bacterial plasmid together, and you want all of the tol gene to be present. You also want to use Ampicillin (Amp) to select for bacteria that take up this plasmid. What enzyme(s) should you use to cut both the plasmid and the genomic DNA? XhoI Plasmid DNA EcoRII XhoI EcoRII BamHI Human genomic DNA XhoI EglI XhoI EcoRII BamHI Amp resistance gene tol gene Ori Unique restriction sites BamHI EglI A. XhoI B. BamHI C. EglI and BamHI D. EcoRII E. BamHI and XhoI

17 Another one: Here is a bacterial plasmid and a piece of human DNA that contains a gene called qt. You want to join the qt gene and the bacterial plasmid together and introduce them into bacteria. You also want to use Ampicillin (Amp) to select for bacteria that take up this plasmid. What enzyme(s) should you use to cut both the plasmid and the genomic DNA? Plasmid DNA XhoI EglI XhoI BamHI Human genomic DNA EglI XhoI EcoRII Ori Amp resistance gene Unique restriction sites EcoRII BamHI EglI qt gene A. XhoI B. BamHI and XhoI C. EglI D. EcoRII E. BamHI and EcoRII

18 An organism that contains DNA from another organism in its genome is called a transgenic organism Thus, a bacterium with a piece of human DNA in it is a transgenic bacterium! A plant engineered to contain a piece of DNA from another organism is also transgenic: we usually call these GMOs 18-18

19 Genetically Modified Foods higher yield improved quality pest or disease resistance tolerance to heat, cold and drought. Plants have been bred for years to specifically yield these desirable qualities Transgenic technology allows the introduction of genes from OTHER organisms into a plant or animal to yield a specific outcome 18-19

20 One example: Insect Resistance Bt corn Normal corn Bacillus thuringiensis (Bt) (a bacterium): Makes a protein that causes paralysis and death to some insects (corn weevil) Use this rather than an applied pesticide, which often kills beneficial insects as well!