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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Lecture, chapter 5 - DNA replication

Monday, December 14, 2009

Today we started chapter 5, on DNA replication.

We discussed some of the generalities of how DNA is duplicated in a cell, and introduced the concept of replication fork. We are listing the enzymes involved in the replisome as we mention each one of their roles in the process.

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Lecture, chapter 4 - Genes, genomes, and DNA

Today we finished chapter 4, on genes, genomes and DNA.

We discussed the ways in which DNA is supercoiled, both in prokaryotes and eukaryotes, similarities and differences between both, and the implications for the functioning of the cell (in terms of replication and gene expression).

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Module 1, Lab 04 - Detection of GM in crops

In this lab we will test corn and soy samples students collected in the Fall to see if they have been genetically modified (if they are Genetically Modified Organisms or GMOs). Our tool of choice for this test will be PCR.

We extracted DNA from corn and soy leaves, as well as from a certified non-GMO seed provided by Bio-Rad. 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.

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Module 1, Lab 03 - PCR of the PV92 Alu insertion locus

Wednesday, December 09, 2009

The goal in this lab to introduce students to the Polymerase Chain Reaction (PCR), the most popular in vitro technique to make copies of target DNA fragments. We extracted DNA from our cheek cells and used it to set up basic PCRs.

Our target is the PV92 Alu insertion locus, located on chromosome 16.

Alu elements are a family of short interspersed repetitive elements (SINEs) that have mobilized throughout primate genomes for the last 65 My, by retrotransposition.

There are more than 500,000 Alu elements per haploid genome in humans (about 5% of our genome). Depending on the insertion point they may be associated with some genetic diseases (e.g.some cases of hemophilia, familial hypercholesterolemia, severe combined immune deficiency, or neurofibromatosis type 1). But in most cases it has no effect on the individual's health.

Some Alu insertions are very recent and polymorphic. The most recent are human specific (HS) and such is the case of PV92. Because the PV92 insertion locus is HS, polymorphic, neutral (invisible for natural selection), and easy to detect, it has been widely used in human genetic population studies, and it has been one of the markers used to support the out-of-Africa hypothesis.

In this lab we will test the presence of 0, 1, or 2 PV92 Alu insertions in our genomes.

The following picture illustrates the possible outcomes of our PCRs:

The sample on lane 1 belongs to an individual with no PV92 Alu insertion, lane 2 to an individual with insertion in both chromosomes, and lane 3 to an individual with an insertion in one chromosome.

What is your genotype like?

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Lecture, chapter 4 - Genes, genomes and DNA

Today we started chapter 4, on genes, genomes and DNA, a discussion on how DNA is organized and how such organization, contrasting prokaryotes and eukaryotes, affects DNA function and replication.

We discussed the different kinds of non-coding DNA (e.g. LINEs, SINEs, introns, satellite DNA, VNTRs...) a few reasons that explain its existence, and a few applications by using them as molecular markers.

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Lecture, chapter 3 - DNA, RNA and Proteins

Today we covered most of chapter 3, on the (very) basic structure and function of DNA, RNA, and Proteins.

Me discussed important concepts like the central dogma of molecular biology, some of the properties that make DNA a key molecule for life (antiparallelism, complementarity), and some of the roles that RNA has, beyond the transfer of information from the nucleus to the cytoplasm.

In our next meeting we'll finish chapter 3, with a basic discussion about protein structure and function.

Reminder: We are meeting tomorrow, Tuesday, in Meyer 128, at 8:00 am, to make up for the class that was cancelled on Monday Nov 30th.

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Lecture, Chapter 3 - DNA, RNA, and Proteins

Today we had our first lecture of the quarter. We went over the syllabus, including a preliminary discussion about the review paper and associated presentation that each student must develop.

We covered most of chapter 3, on DNA, RNA, and proteins. We discussed about the most basic structure of nucleic acids and some of the differences between DNA and RNA.

Reminder: We will have lecture on Tuesday Dec 8 at 8:00 a.m. to make up for the lecture we didn't have on Monday Nov 30.

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