Lecture, chapter 10 - Transcriptional gene regulation in eukaryotes

We finished covering chapter 10, on transcriptional gene regulation in eukaryotes. We devoted special attention to gene silencing via formation of heterochromatin, a process in which histone acetylation and cytosine methylation are key processes.

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Lab 11 - Polyacrylamide Gel Electrophoreses (PAGEs)

Friday, October 23, 2009

Today we did a new kind of electrophoresis, or you could say that we did two. Polyacrylamide Gel Electrophoresis (PAGE) is a process that uses the same principle of agarose gel electrophoresis, but it uses a polyacrylamide gel, a thinner, more expensive kind of gel that provides a higher resolution than its agarose counterpart.

Specifically we used it to run protein samples and we did it in two ways. We ran a native gel, in which the proteins migrate at different rates depending on their size (molecular weight), tertiary structure, and charge. We also ran a denaturing gel, in which the proteins are denatured with high temperature and kept denatured by SDS contained in the electrophoresis buffer, so their rate of migration through the gel depends exclusively on size.

The goal was to estimate the size of the green fluorescent protein (GFP) by comparing its migration through each gel with the migration of a molecular weight ruler (a "protein ladder") loaded onto the same gel.

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Lab 10 - Protein Quantitation (Bradford Protein Assay)

From the Quick Start™ Bradford Protein Assay

instruction manual (BioRad)

Today we did a protein quantitation using BioRad's Quick Start™ Bradford Protein Assay, a method in which a dye reagent is used (Coomassie Brilliant Blue) to bind to proteins and measure its absorbance. The more concentrated the protein it binds, the darker the blue resultant color, and the greater the absorbance at 595 nm.

Two standard proteins are used, bovine serum albumin (BSA) and gamma-globulin, to generate absorbance vs. protein concentration curves and then interpolate the absorbance of problem samples to estimate their concentration. The protein samples obtained from the Hydrophobic Interaction Chromatography (HIC) are used as problem samples.

This method is applied when researchers in proteomics discover a new protein and are trying to gather information about it. In our case, we "discovered" GFP, although we wouldn't have a name yet, had it been a truly newly discovered protein.

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Lectures, chapters 9 & 10 - Transcriptional gene regulation in prokaryotes and in eukaryotes

Yesterday we covered most of the relevant section in chapter 9, on transcriptional gene regulation in prokaryotes. Today we finished and started chapter 10, on transcriptional gene regulation in eukaryotes.

Transcriptional gene regulation in prokaryotes covers the basics of gene regulation, including the concepts of global and specific regulation, activators and repressors, and regulation at transcription termination.

Transcription gene regulation in eukaryotes adds layers of complexity, like the role played by specific transcription factors, accessibility to DNA condensed in nucleosomes, and the role played by a mediator complex during gene activation.

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Lab 09 - DNA sequencing

Friday Oct 16 2009

Today we ran gel electrophoreses to confirm if the plasmid extraction (lab 8) was successful and if we had the GAPC gene (from Arabidopsis) insert. In some cases we did.

Using the samples that had the insert we mixed the miniprep DNA with forward and reverse sequencing primers (pJET SEQ F and pJET SEQ R), and put them in a 96-well plate. The plate will be shipped to the DOE Joint Genome Institute (JGI) to be sequenced as part of their Sequencing Training Program (STR). The results should be in in two weeks, ready to be used in the bioinformatics labs

The report from lab 9 will be merged with the report of lab 15 (Bioinformatics)

While the gels were running we discussed the DNA sequencing technique most commonly used: Dye-terminator sequencing, a modification of Sanger's chain termination sequencing protocol, which allowed the automation of the sequencing process.

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Lab 08 - Ligation & Transformation

Today we almost finished lab 08, the ligation and transformation module (ligation of the Arabidopsis GAPC gene into the pJet1.2 plasmid; transformation of E. coli).

We did a plasmid DNA extraction (minipreps) from the bacterial cultures we did last week, and initiated a restriction enzyme digestion with BglII to confirm if we have plasmid DNA and the insert we are interested in. Tomorrow we will run an agarose gel electrophoresis to confirm the results.

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Lecture, chapters 7 and 9 - Protein structure and function & Gene regulation in prokaryotes

Today we finished chapter 7, on protein structure and function. We discussed the main structural motifs found in DNA-binding proteins, and talked about protein denaturation.

We also started covering chapter 9, on gene regulation on prokaryotes, focusing on regulation at the transcription level. We discussed the importance of gene regulation and some of the key players involved in the process.

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Lecture, chapter 7 - Protein structure and function

Today we covered most of chapter 7, on protein structure and function. We further discussed the secondary structure of proteins (α-helices and β-sheets), and we talked about the tertiary and quaternary structures as well.

In terms of function we mainly discussed how DNA-binding proteins can read the information in the double helix without breaking the hydrogen bonds between bases.

Reminder: The first draft of the review paper is due tomorrow at noon. Send me an electronic file (preferably a Word document) before then.

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Labs 07 and 08

Friday October 09 2009

Lab 08 - Ligation and transformation

Students used bacterial colonies from the LB/Ampicillin agar plates incubated yesterday to inoculate LB/Ampicillin broth media. The broth media will be incubated for approx. 24 hours at 37ºC in a shaking water bath at approx. 200 rpm.

Lab 07 - Protein purification by chromatography

Students used the bacterial cultures inoculated yesterday to isolate Green Fluorescent Protein (GFP) by the process of Hydrophobic Interaction Chromatography (HIC).

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Labs 06, 07, and 08

Thursday October 08 2009

Lab 06 - pGLO bacterial transformation

Students analyzed the results of the bacterial cultures in LB, LB/Ampicillin, and LB/Ampicillin/Arabinose agar plates. A colony in the latter will be used to start lab 07.

Lab 07 - Protein purification by chromatography

Students used a colony from the LB/Ampicillin/Arabinose agar plates generated in lab 06 to inoculate an LB/Ampicillin/Arabinose broth tube. The broth was incubated at 37ºC for approx. 24 hours in a shaking water bath at approx. 200 rpm.

Lab 08 - Ligation and genetic transformation

Students prepared competent cells (E. coli) and transformed them with the pJet1.2 plasmid that has the GAPC gene (from Arabidopsis) insert.

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Lecture, chapter 7 - Proteins

We finished chapter 6, on transcription of genes, and went ahead and started chapter 7, on protein structure and function.

We talked about amino acids, the monomers that make up polypeptides, and how they are organized in four levels of structure. We also made the distinction between a polypeptide and a protein. We took a closer look to the primary and secondary structures of polypeptides (and proteins).

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Lecture, chapter 6 - Transcription of genes

Today we covered most of chapter 6, on transcription of genes.

We compared some details of transcription in prokaryotes and eukaryotes, including the kind of RNA polymerases involved in each one, the transcription factors involved in eukaryotic transcription, and some of the components of regulatory DNA.

Reminder: Next Wednesday (Oct 14) is the due date for the first draft of the review paper. The more advanced the paper the more feedback I'll be able to give you!

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Lab 06 - Bacterial genetic transformation with pGLO Lab 08 - Ligation and transformation (gene GAPC)

We performed the genetic transformation of E. coli cultures with BioRad's pGLO™ plasmid, engineered to contain the green fluorescent protein (GFP) gene , originally isolated from the crystal jelly Aequorea victoria.

We also performed the ligation of our GAPC gene (obtained in lab 05) with BioRad's pJet1.2 plasmid. In the next lab we will genetically transform bacteria, just as we did earlier today.

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Lab 05 - GAPDH Nested PCR

Thursday, October 1st 2009

We ran the gels for the nested PCRs we performed last week.

Due to failure of starter bacterial cultures we couldn't start lab 6, on bacterial genetic transformation, but we did talk about the main topics the lab is related to: Gene regulation, antibiotic resistance, and genetic transformation.

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Exam 1

Today we had our first partial exam.

Results and stats:

(click image for full size view)

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Lecture, Chapter 6 - Transcription

After finishing chapter 5, on the few details that differ in eukaryotic DNA replication compared to that in prokaryotes, we started chapter 6, on transcription.

We talked about the basics of the synthesis of transcripts, and got acquainted with some new terms like cistron, open reading frame (ORF), monocistronic and polycistronic mRNAs, and operon.

Tomorrow: Exam 1...!!!

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Lab 05 - GAPDH Nested PCR

Arabidopsis thaliana

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Today we started the exercise in which students will learn the basics of a nested PCR. We will work with the gene that encodes one of the GAPDH isomers, GAPC, in Arabidopsis thaliana, the model organism of plants. Some people call it "the fruit-fly of plants".

GAPDH is an enzyme in charge of catalyzing one of the reactions in glycolysis. There are several nuclear genes that encode GAPDH isomers (proteins with different amino acid sequences but with the same function), and we are targeting the gene GAPC in the A. thaliana genome. We ran a first round of PCR, with our initial primers, and tomorrow, Friday, we will run the second run, with the nested primers.

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Lecture, Chapter 5 - DNA replication

We continued covering chapter 5, on DNA replication. We finished talking about DNA replication in prokaryotes, topic in which students should have an understanding of the replication fork, including the functioning of all the enzymes involved in the process.

We started talking about DNA replication in eukaryotes, organisms in which some differences are found, mainly because of the linearity of chromosomes. In prokaryotes chromosomes are circular.

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Lecture, Chapters 4 and 5 - Genes, Genomes, and DNA & DNA Replication

Today we covered the end of chapter 4, focusing mainly on the mechanisms prokaryotes and eukaryotes use to supercoil their DNA.

Then we started with chapter 5, on DNA replication. We introduced the concept of replication fork and went over some of the issues the cell has to solve in order to get supercoiled DNA to replicate.

Reminder: We are in week 3 and students should be meeting with me to decide the topic of the review paper.

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Lab 04 - Detection of genetic modification in crops

Friday Sep 18 2009

We started the exercise in which we will test corn and soy samples students brought to the lab to see if they have been genetically modified (if they are Genetically Modified Organisms or GMOs).

We extracted DNA from corn and soy leaves, as well as from a certified non-GMO seed provided by Bio-Rad with the kit. We set up PCRs using primers that will amplify de 35S promoter of the cauliflower mosaic virus (CaMV 35S) and the nopaline synthase (NOS) terminator of Agrobacterium tumefaciens, which are present in about 85% of all modified crops in the U.S. As a positive control for the presence of DNA, we also used primers that amplify the photosystem II chloroplast gene, which should be present in all plants, regardless of genetic modification.

Next week we will run an agarose gel electrophoresis to confirm the results.

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