Yesterday s Picture UNIT 3B

Transcription

1 Warm-Up Plasmids are circular pieces of DNA which bacterial cells are able to take up from the environment, then replicate and transcribe. Eukaryotic cells, by contrast, contain large, linear (non-circular) DNA molecules called chromosomes. Predict which type of DNA (plasmid or chromosome) would require greater activity of topoisomerase during replication, and justify your prediction.

2 Yesterday s Picture

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4 Bacteria (prokaryotes) naturally have large circular chromosomes, but can also take up small circular plasmids (a process called transformation).

5 Bacteria (prokaryotes) naturally have large circular chromosomes, but can also take up small circular plasmids (a process called transformation). Artificial plasmids called vectors are designed for bacteria to express specific genes (make specific proteins).

6 Bacteria (prokaryotes) naturally have large circular chromosomes, but can also take up small circular plasmids (a process called transformation). Artificial plasmids called vectors are designed for bacteria to express specific genes (make specific proteins). Vectors contain: Your gene of interest A promoter which recruits RNA polymerase when activated. promoter RNAP your gene of interest RNA DNA

7 CTQ #1 Scientists are able to purify mrna from the human insulin gene in vitro (in a test tube using enzymes). They then use the purified RNA to synthesize DNA. Finally, they insert the synthesized DNA into a linearized (cut open) plasmid. State the THREE enzymes they must use during this process.

8 Vectors contain: Your gene of interest A promoter where RNAP initiates transcription when recruited. An antibiotic-resistance gene

9 Vectors contain: Your gene of interest A promoter where RNAP initiates transcription when recruited. An antibiotic-resistance gene First, scientists transform bacteria and spread the bacteria on an agar (food) plate.

10 Vectors contain: Your gene of interest A promoter where RNAP initiates transcription when recruited. An antibiotic-resistance gene First, scientists transform bacteria and spread the bacteria on an agar (food) plate. Next, the plate is treated with an antibiotic (such as ampicillin) for which the vector encodes resistance. (Non-transformed bacteria die). Transformed bacteria Non-transformed bacteria

11 Vectors contain: Your gene of interest A promoter where RNAP initiates transcription when recruited. An antibiotic-resistance gene First, scientists transform bacteria and spread the bacteria on an agar (food) plate. Next, the plate is treated with an antibiotic (such as ampicillin) for which the vector encodes resistance. (Non-transformed bacteria die). No ampillicin smear of lots of bacteria Added ampicillin - only transformed bacteria grow

12 Vectors contain: Your gene of interest A promoter where RNAP initiates transcription when recruited. An antibiotic-resistance gene First, scientists transform bacteria and spread the bacteria on an agar (food) plate. RNA Next, the plate is treated with an antibiotic (such as ampicillin) for which the vector encodes resistance. (Non-transformed bacteria die). Arabinose activator RNAP Finally, an activator of the promoter (such as arabinose) is added. promoter your gene of interest

13 Vectors contain: Your gene of interest A promoter where RNAP initiates transcription when recruited. An antibiotic-resistance gene First, scientists transform bacteria and spread the bacteria on an agar (food) plate. Next, the plate is treated with an antibiotic (such as ampicillin) for which the vector encodes resistance. (Non-transformed bacteria die). Finally, an activator of the promoter (such as arabinose) is added. No arabinose no GFP genes activated Added arabinose GFP genes activated

14 Vectors contain: Your gene of interest A promoter where RNAP initiates transcription when recruited. An antibiotic-resistance gene GFP protein First, scientists transform bacteria and spread the bacteria on an agar (food) plate. Next, the plate is treated with an antibiotic (such as ampicillin) for which the vector encodes resistance. (Non-transformed bacteria die). Finally, an activator of the promoter (such as arabinose) is added. The gene of interest (commonly Green Fluorescent Protein or GFP) is activated. GFP fluorescence phenotype

15 The gene of interest (commonly Green Fluorescent Protein or GFP) is activated. Any protein which needs to be synthetically made (such as insulin for diabetes patients) can be inserted instead of GFP.

16 CTQ #2 Kanamycin is an antibiotic which is used in transformation experiments. Explain why discrete colonies form on kanamycintreated plates following a transformation experiment using a plasmid encoding kanamycin resistance.

17 Closure Describe the difference, and the importance, between ampicillin and arabinose in a bacterial transformation experiment.