1. BIOL 417 – Week 2, Day 2 Prepared in large part byPrepared in large part by –Celithelma Garcia –Mijin Sohn and –Flora Hwee of PGM 2000 Revised 2012, SBSharpRevised 2012, SBSharp

2. Recap 1. Modification of the Insert: When the cDNA is made, overhangs may remain. When the cDNA is made, overhangs may remain. 3’CCCCC---------------------------TTTT 5’ 3’CCCCC---------------------------TTTT 5’ 5’ GGGG---------------------------AA 3’ 5’ GGGG---------------------------AA 3’ Add T4 polymerase and dNTPs: 3’ CCCC ---------------------------TTTT 5’ 3’ CCCC ---------------------------TTTT 5’ 5’ GGGG---------------------------AAAA3’ 5’ GGGG---------------------------AAAA3’

3. Recap (continued ) 2. PstI adaptors were added in excess to polished cDNA to create sticky ends. 3. Ligase was added to connect the adaptor and cDNA, thus completing the insert preparation. 4. Vector had been opened with PstI.

4. What’s next? I. Forming recombinant plasmid DNA, i.e., the “construct” (in vitro) II. Transformation of ligation reaction products into E. coli bacteria (in vitro  in vivo) At least some of the ligation products are expected to be vector + insert ligated together. At least some of the ligation products are expected to be vector + insert ligated together. III. Selection of bacteria that have taken up plasmid +/- insert (in vivo)

5. I. Forming the Recombinant Construct The insert is added to the vector and ligase is also added The insert is added to the vector and ligase is also added 3’ HO-ACGTC-----CCCCC--------------TTTTT------G-OH 5’ 5’ HO-G-----GGGGG-------------AAAAA-----CTGCA-OH 3’ 5’ HO-G-----GGGGG-------------AAAAA-----CTGCA-OH 3’ + 3’ HO-ACGTC-----------------------------------------------G-PO 4 5’ 5’ 4 OP-G------------------------------------------------CTGCA-OH 3’ 5’ 4 OP-G------------------------------------------------CTGCA-OH 3’ +ligase

6. Diagram the ligation product of the two molecules on the prior slide.

7. I. Recombinant (continued) Ligase connects where there is a -PO 4 -2 at 5’ end and –OH at adjacent 3’ end.Ligase connects where there is a -PO 4 -2 at 5’ end and –OH at adjacent 3’ end. Because the PstI adaptor’s 5’ sticky-end has a chemically synthesized –OH group rather than -PO 4 -2, ligase cannot ligate the sticky end of the insert to the 3’ end –OH of the vector.Because the PstI adaptor’s 5’ sticky-end has a chemically synthesized –OH group rather than -PO 4 -2, ligase cannot ligate the sticky end of the insert to the 3’ end –OH of the vector. But, the construct remains together due to the non-covalent H-bonds at the base pairs throughout the length of the insert.But, the construct remains together due to the non-covalent H-bonds at the base pairs throughout the length of the insert.

8. II.Transformation II.Transformation Putting construct into bacteria for replication Definition: The process of introducing the products of a ligation reaction into E. coli for replication to high copy number.Definition: The process of introducing the products of a ligation reaction into E. coli for replication to high copy number.

9. Transformation The basic methodology is:The basic methodology is: –Incubate E.coli in CaCl 2 solution to make them “competent”. –Add ligation reaction to the competent E. coli. –CaCl 2 precipitates DNA into clumps and increases DNA adsorption to cell membrane. –Heat shock the bacteria/DNA mixture to facilitate DNA uptake by the competent bacteria. Note: This same methodology is used with a previously made plasmid construct in order to grow up more of it.Note: This same methodology is used with a previously made plasmid construct in order to grow up more of it.

10. II. Transformation Possibilities of DNA uptake by bacteria (1). Some bacteria take up reclosed vector.Some bacteria take up reclosed vector. 1. Vector Bacterial DNA

11. Some bacteria take up only the insert.Some bacteria take up only the insert. Bacterial DNA Insert II. Transformation Possibilities of DNA uptake by bacteria (2).

12. Some bacteria take up the product of interest, the construct you want.Some bacteria take up the product of interest, the construct you want. Bacterial DNA Vector + Insert (recombinant) II. Transformation Possibilities of DNA uptake by bacteria (3).

13. Some bacteria will contain a construct with a shorter or a longer insert than the one you expect.Some bacteria will contain a construct with a shorter or a longer insert than the one you expect. Bacterial DNA Vector + Shorter Insert (recombinant) II. Transformation Possibilities of DNA uptake by bacteria (4).

14. IV. Possibilities in Attempting to Transform Bacteria (5) Most bacteria will fail to take up any ligation products.Most bacteria will fail to take up any ligation products. Bacterial DNA

15. Possibilities in Attempting to Transform Bacteria (summary) There are more transformation possibilities; only five are presentedThere are more transformation possibilities; only five are presented 1 2 345 All types and more are in the same transformation reaction. We must distinguish bacteria that have taken up plasmid. Later, we must distinguish the product of interest from other transformation products.

16. II. Summary: Putting construct into bacteria for replication (to get enough DNA to work with!) E. coli that have been made competent can take up naked DNA.E. coli that have been made competent can take up naked DNA. Only 1 in a 1000 competent E. coli will take up DNA.Only 1 in a 1000 competent E. coli will take up DNA. Only a fraction of that fraction will take up the recombinant construct you want, because the construct you want is only a fraction of possible ligation products.Only a fraction of that fraction will take up the recombinant construct you want, because the construct you want is only a fraction of possible ligation products.

17. Realities of cDNA construction. Some cDNAs will form that are not full length.Some cDNAs will form that are not full length. Some vectors will reclose on themselves with no cDNA insert.Some vectors will reclose on themselves with no cDNA insert. Some vector molecules may never have opened!Some vector molecules may never have opened! Nothing ever works the way you want it to 100% of the time.

18. III. Selection of bacteria that have taken up plasmid. How can you discriminate between bacteria that have taken up plasmid (either +/- insert) and the other 99.9% of the bacteria?How can you discriminate between bacteria that have taken up plasmid (either +/- insert) and the other 99.9% of the bacteria? –Take advantage of the drug resistance gene in the plasmid vector. Amp r = gene that codes for an enzyme that breaks down ampicillin, a drug that stops bacterial cell division.Amp r = gene that codes for an enzyme that breaks down ampicillin, a drug that stops bacterial cell division. Because the plasmid includes a gene for Amp r, the presence of the plasmid gives (confers) ampicillin resistance to the bacterium.Because the plasmid includes a gene for Amp r, the presence of the plasmid gives (confers) ampicillin resistance to the bacterium. Include ampicillin in the nutrient agar in the bacterial culture plate.Include ampicillin in the nutrient agar in the bacterial culture plate.

19. Vector

20. IV. Selection of Desired Recombinant Bacteria (cont.) Amp - Transformed bacteria <1/1000 have plasmid. There are many thousands of bacteria in the tube. Amp+ -Contains the Amp r gene Transfer to a medium with Amp Transfer to a medium without ampicillin

21. Notice the colonies. The bacteria in each colony contain plasmids. What must be present in the agar to ensure the presence of plasmids in the bacteria that grow?

22. Summary of selection. All bacteria can grow on Amp - plate.All bacteria can grow on Amp - plate. Only those bacteria carrying plasmids with an Amp R gene will grow on Amp + medium and form colonies.Only those bacteria carrying plasmids with an Amp R gene will grow on Amp + medium and form colonies. –Cells containing only the insert will not grow on Amp + medium.

23. Characteristics of a Good Vector 1Origin of Replication: allows the vector to replicate independently 2 Replicates to high copy number 3 Small genome that can be isolated easily 4 Unique restriction enzyme sites for DNA insertion 5 A way to discriminate its presence in the bacterium, e.g. ampicillin resistance 6 Optional: A way to discriminate the presence of an insert, e.g. breakdown of X-gal by  - galactosidase (next time)