Connecting the last few labs

The last several labs have been connected and here's a little summary to remind you how they are interconnected:

• Labs 7-8: Bacterial genetic transformation with pGLO plasmid
• Labs 9-10: Protein (GFP) purification by chromatography
• Labs 11-12: Bradford protein (GFP) determinations
• Lab 13: Native Protein PAGE
• Lab 14: Denaturing Protein PAGE

When we transformed our E. coli with the pGLO plasmid, containing the gene for the Green Fluorescent Protein (GFP) we were also producing the material we were going to use in the next several labs. We transformed the bacteria, made them produce our GFP, then purified that GFP and now we are determining how much of it there is and how big the polypetide is.
This process is analogous to that in the industry to produce a protein for commercial applications asn also abalogous to a research process when a gene for a new protein is being discovered or studied.

Dont think of these few labs as independent ones, but as steps of a single longer lab.

Labs 11-14 can be summarized in a single lab report. Here are the questions for such report:

1. Why do we make our absorbance measurements at 595 nm?
2. When you heat up DNA or protein samples up to 95ºC they get denatured. Specifically what happens to DNA and proteins? In other words what is DNA denaturation and what is protein denaturation?
3. When estimating protein size would you rely more on the native of the denaturing gel results? Why?
4. Some groups had more than one protein (more than just GFP) in their gels. HOw do you explain this?
   

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LAB - Denaturing Protein PAGE

Today we ran another protein PAGE, but in this case it was a denaturing gel as opposed to a native one. We denatured both, our GFP samples and our standards by heating the samples at 95º C for 2-5 min.

We will compare the results of the native and denaturing gels in terms of the estimates that they provide of the size (molecular weight in Daltons)of GFP.

Since we are one lab ahead, and the next couple of labs must be done back to back, we will not be having a lab session tomorrow Friday.

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DNA replication machinery

DNA replication "fork" and enzymes involved in the DNA replicaiton process.
This pic summarizes this class. Click on it for a full size image.

Yesterday we ahd a little change in the class dynamic and it served also as a little experiment. Instead of me lecturing all the time, students were given time to discuss the class topics in bewteen them, and then were asked to present them to their peers.

Several teams of two students explained DNA replication machinery topics, such as templated polymerization, semiconservative replication, replication "fork", Okazaki fragments, etc.

IMPORTANT: Even though we didn't cover all the mateiral up to page 281, these topics should be read and will be included in the next exam on Nov. 4th! These include proofreading mechanisms, and proteins invloved in the replication process.

The pic at the top of this blog entry is a good summary of what we studied in class and what you shoud read to complement what was covered.

Reading for next class: Pages 295-306


Quiz #10 Q&As

1. What is mutation rate?
Rate at which mutations accumulate in a genome

2. How can you measure mutation rate directly?
Sequencing and comparing genomes of an ancestor and some if its descendants

3. How can you measure mutation rate indirectly?
Comparing DNA sequences, especially introns, of closely relates species

4. What is a replication "fork"?
Active region of replication in a parental DNA double helix observed as a 'Y' shaped structure as a result of the separation of DNA strands

5. What is a ‘proofreading’ mechanism in the context of DNA replication?
Chemical mechanisms in which DNA Polymerase "double-checks" that polymerased bases are the right ones (complementary to those in the template strand)


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Genetic stability and mutation

We discussed the importance of mutation. Even though mutation results from mistakes in the DNA replication system, which it's supposed to be perfect, it is the force that drives organic evolution and the reason that life is as diverse as it is.

Rate of mutation is extremely low compared to the rate of success of DNA replication, in part because there are several checks during the pocess and even after that tere are mechanisms of DNA repair. For a mutation to occur several "security gates" must be passed. But since populations tend to be large mutations can have an impact over evolutionary time.

It has been estimated (according to our text book) that the average rate of mutation is relatively constant across the tree of life: 1 mutation/10 exp9 nucleotides/cell generation (prokaryotes) and 1 mutation/10 exp9 nucleotides/cell division (eukaryotes).

Mutation is te single force behind evolution and cancer. I can be advantageous, or it can be deletereous.


Quiz #9 Q&As

1. Mention 3 kinds of mutations that promote genome evolution
Point mutation, translocations, deletions, insertions, duplications, inversions...

2. What are ‘regions of synteny’?
Genomic regions with the same genes in the same order in different species

3. Mention a way in which multispecies genome comparisons can be used (a question you can answer with them)
Inferring function of DNA sequences, inferring evolutionary relationships (phylogeny), determining genetic distance...

4. What are Human Accelerated Regions (HARs)?
Genomic regions that are highly conserved accross species but have a high (accelerated) rate of mutation in humans

5. What enzyme is responsible for adding nucleotides to a DNA strand during replication using the other DNA strand as a template?
DNA Polymerase

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