Exam 2

Today we had our second exam.

Statistics:

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Lab 12 - Polyacrylamide Gel Electrophoreses (PAGEs) of GFP

Today we discussed a new kind of electrophoresis: 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 were supposed to use it to run protein samples and in two ways.

• A native gel, in which the proteins migrate at different rates depending on their size (molecular weight), tertiary structure, and charge.
• 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. Unfortunately we didn't have the reagent to stain the gels (coomasie), so we didn't actually run the gels, although we did talk about the process.

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Lab 11 - Protein quantitation

Wednesday, February 3, 2010

Today we did a protein quantitation using BioRad's Quick Start™ Bradford Protein Assay, a method in which a dye reagent is used (Bradford reagent, based on Brilliant Blue G-250) to bind to proteins (causing the dye reagent to change to a different color) 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 relative standard proteins are used, bovine serum albumin (BSA)and bovine gamma-globulin (BGG), 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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Lecture, Chapter - Gene regulation at the RNA level

Today we covered chapter 11, on gene regulation at the RNA level.

We discussed mechanisms of gene regulation between transcription and translation:

• Rate of degradation of mRNA
• Modification of mRNA
• Control of translation by RNA binding proteins
• Anti-sense RNA
• Ribosome alteration (preferential translation of mRNAs)
• RNA interference (RNAi)
• Micro RNA (miRNA)
• Riboswitches

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Lecture, chapter 10 - Gene regulation in eukaryotes

Today we finished chapter 10, on gene regulation in eukaryotes.

We discussed the role of DNA looping and insulators in ensuring that enhancers have an effect only on associated genes.

Then we discussed how a cell deals with the fact that much of its DNA is found as heterochromatin, making it inaccessible for RNA polymerase, and how DNA must be turned into euchromatin to be able to be expressed... or how euchromatin can be turned into heterochromatin to turn genes off (histone acetylation/deacetylation, DNA methylation/demethylation).

We also introduced the concepts of gene silencing and genetic imprinting

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Lab 7b - Smal scale plasmid purification (pJet1.2 with GAPC gene insertLab 13 Small scale plasmid purification (pGLO plasmid)

Today we used the bacteria we transformed (and cloned) with the pGLO and pJet1.2 plasmids to perform small scale plasmid DNA purifications (minipreps) and isolate the plasmids.

Then we performed restriction enzyme digestions, RED, using the restriction enzymes Ec0RI and HindIII (see restriction map) for the pGLO plasmid, and BglII for the pJet1.2 plasmid.

The idea was to isolate the GFP gene in a fragment, in the case of pGLO, and to confirm the success of the ligation procedure in the case of pJet1.2.

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Lecture, chapter 9 - Gene regulation in prokaryotesLecture, chapter 10 - Gene regulation in eukaryotes

Today we finished chapter 9, on gene regulation in prokaryotes, and started chapter 10, on gene regulation in eukaryotes.

We discussed the ways in which a protein can be a promoter for some genes and at the same time a repressor for others, and the role an inducer may have in changing its tertiary structure.

We also compared the action of global vs. specific regulators and introduced the concept of regulon.

Finally, we discussed the anti-termination control mechanism for regulation of gene expression.

We talked about the reasons control of gene expression in eukaryotes is more complex than in prokaryotes (supercoiling, presence of a nuclear envelope, cell differentiation in multicellular ones, developmental reasons, etc.).

We describe how the transcription apparatus is assembled and the role the mediator complex plays in facilitating its interaction with specific transcription factors.

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Lecture, chapter 10 -Gene regulation in prokaryotes

Today we stated chapter 10, on gene regulation in prokaryotes.

We discussed the importance of having gene regulation mechanisms in place for the correct functioning of prokaryotic cells, and why it is more common to find such mechanisms acting upon transcription than other cellular processes.

We also discussed the difference between positive and negative regulation, the role activators and repressors play in such processes, and how they are affected by the presence of inducers in the environment.

The basics of the operon model of gene regulation were also covered.

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Lab 10 - Protein purification(GFP) through Hydrophobic Interaction Chromatography (HIC)

Today we performed a Hydrophobic Interaction Chromatography (HIC) to separate the green fluorescent protein (GFP), produced in our bacterial cultures, from other proteins commonly found in bacteria.

A sample of bacteria was concentrated and then resuspended in a solution in which they were lysed. The high salt solution, containing all the proteins found in the bacteria, was then passed through a hydrophobic interaction column where molecules of the naturally hydrophobic GFP bound to the hydrophobic beads. The high salt solution increased the hydrophobicity of GFP by further exposing its hydrophobic residues.

A series of washes with buffers of decreasing salinity allows proteins with various levels of hydrophobicity to gradually unbind from the beads and be collected in a test tube. By switching collection tubes each time a buffer is added, different proteins can be collected. One of them was GFP and the tube in which it was collected should glow.

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Lab 07 - Ligation and transformation (GAPC gene from Arabidopsis and pJet1.2 plasmid)

Wednesday, January 20, 2010

Today we used the purified PCR product from the nested PCR lab (GAPC gene from Arabidopsis) to genetically transform E. coli.

The lab was divided in three main steps

• Preparation of competent cells
• Ligation
• Genetic transformation

During most of the lab students manipulated bacteria to make them competent (i.e. get them ready to uptake extracellular DNA). Once this was achieved, the GAPC gene from Arabidopsis, obtained via nested PCR, was ligated to the pJet1.2 plasmid.

The plasmid was then used to genetically transform E. coli that were spread on LB agar/Amp/IPTG plates and incubated.

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Lecture, chapter 7 - Protein structure and function

Today we continued with the chapter in protein structure and function.

We discussed the most common shapes found in the secondary structure of proteins, α-helices and β-sheets. Me mentioned the typical ways in which they refold to obtain their tertiary structure.

We talked how can proteins "read" DNA information without separating the strands in the double helix and the most common DNA-binding motifs found (helix-turn helix, helix-loop-helix, leucine zipper, zinc finger).

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Module 3, Lab 9 - Genetic transformation of E. coli with the pGLO plasmid

Aequorea victoria, original source of the green fluorescent protein (GFP)
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First lab in module 3. Today we used the pGLO plasmid to genetically transform E. coli.

pGLO is a plasmid that has been engineered to contain and express the Green Fluorescent Protein (GFP) gene, originally isolated from the jelly Aequorea victoria. GFP produces a green fluorescence when excited by blue or UV light.

In order to make the GFP gene a functional one it has been engineered so the sugar arabinose triggers the production of the protein. The genes in the arabinose operon (araB, araA, and araD) have been replaced by the GFP gene. Such genes encode proteins that break down arabinose when it is present in the environment, so they are expressed only if this is the case. The activating mechanism has been left intact, so in the engineered operon the presence of arabinose turns on the GFP gene and therefore GFP is produced.

Another feature of the pGLO plasmid is the presence of the beta-lactamase gene, which provides resistance against the antibiotic ampicillin.

The bacteria were transformed through the heat shock technique, and then plated on LB agar plates containing:

• Just LB (lysogeny broth)
• LB and ampicillin
• LB, ampicillin and arabinose

Plates are being incubated for 24 hours at 37ºC.

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Lecture, chapter 6 - Transcription in eukaryotesLecture, chapter 7 - Protein structure and function

Today we finished chapter 6, on transcription, specifically in eukaryotes.

We discussed the mechanism in which general and specific transcription factors aid RNA polymerase II in initiating transcription of protein-coding genes in eukaryotes. We also discussed the role of enhancer regions as sequences recognized by specific transcription factors and the role of DNA looping to allow the interaction of transcription factors that bind to the DNA far away from the gene (or transcription unit).

We also started chapter 7 on protein structure and function. We covered the characteristics of amino acids and then we started discussing the levels of structure in polymers, specifically polypeptides.

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Exam 01

Today we had our first exam of the quarter.

Statistics:

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Module 2, Lab 06c - Nested PCR of the GAPC gene of Arabidopsis thaliana

Thursday, January 7, 2010

Today we ran a gel electrophoresis with the second round PCRs of the Arabidopsis GAPC gene. Once we confirmed the results of the PCRs we purified the samples to get them ready for ligation and transformation in the next labs.

The starter E. coli agar plates that we were supposed to use to begin lab 9 did not work and will be repeated. On Monday we'll meet to do the genetic transformation of E. coli with the pGLO plasmid, on Tuesday we will inoculate liquid media and on Wednesday we will do lab 10: purification of the green fluorescent protein (GFP) through hydrophobic interaction chromatography.

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Module 2, Lab 06b - Nested PCR of the GAPC gene of Arabidopsis thaliana (second round)

Today, under the direction of Dr. Dennis DeLuca (since I was out of town) students set the second round of PCRs to amplify the GAPC gene of Arabidopsis.

Primers of the first round were inactivated with the use of an exonuclease, and the exonuclease was inactivated through an incubation at 8oºC.

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Lecture, chapter 6 - Transcription in prokaryotes

Today we started the chapter on transcription.

We discussed the basics of transcription in prokaryotes, including some terminology specific to the transcription process and features necessary for it (e.g. roles of promoter and terminator regions)

Reminders:

• Labs 2 and 3 are due today
• We have our first exam on Friday

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Lecture, chapter 5 - DNA replication

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Today we finished chapter 5, on DNA replication. We went into details of how the lagging strand is synthesized, forming the Okazaki fragments, thanks to DNA looping, which allows for it's synthesis even though the helicase and polymerase III-dimer complex travels in the same direction of the replication fork. We added how the RNA primers are degraded and the Okazaki fragments are joined, by the action of ribonuclease H, DNA polymerase I, and DNA ligase.

We then discussed how DNA transcription proceeds in eukaryotes, what's the role of telomerase in reparing telomeres after each round of replication, and the differences between the action of polymerases in prokaryotes and eukaryotes.

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Module 2, Lab 06a - Nested PCR of the GAPC gene of Arabidopsis thaliana

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 on Wednesday after Christmas break we will run the second round, with the nested primers.

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Module 1, Lab 05 - Size Exclusion Chromatography (SEC)

Today we did the agarose gel electrophoresis for lab 04 (detection of genetic modification in crops), using 2% agarose gels.

Then we did lab 05, size exclusion chromatography (SEC), in which a sample mix of hemoglobin and vitamin B12 were separated by size by column chromatography.

Once the procedure was finished we discussed agarose gel interpretation, specifically for labs 02 and 03.

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