Exam 3 (final)

Stats for the final exam (scores out of 150 points):

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Lecture - Molecular techniques

Today we had a discussion in which we summarized some of the most important techniques that are used to study proteins and nucleic acids. Topics that were covered:

• Protein purification
• Denaturing PolyAcrylamide Gel Electrophoresis (SDS-PAGE)
• Two-dimensional electrophoresis
• PCR
• PCR in disease diagnosis
• DNA sequencing
• Chain termination method (Sanger method - manual and automated)
• Shotgun sequencing
• Pyrosequencing
• Next-generation sequencing
• DNA typing

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6th ONU Intro Molecular Biology SymposiumSecond session

Department of Biological and
Allied Health Sciences
Mathile Center 108
February 14 & 17, 2011

The ONU Intro Molecular Biology Symposium takes place every Fall and Winter quarters, as part of the Introduction to Molecular Biology (Biol 217) class. Speakers are students registered in the class, who throughout the quarter have written a review paper on molecular biology-related topics.

Click on pic for a full size image

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Lecture, chapter 12 - Processing of RNA

Today we talked about how introns are removed by using a combination of small nuclear RNA (snRNA) and proteins called small nuclear ribonucleoproteins (snRNP, a.k.a. "snurps").

Then we discussed alternative RNA splicing mechanisms:

• Alternative promoter selection
• Alternative tail site selection
• Alternative splicing by exon cassette selection
• Trans-splicing (rare)

On Friday: Molecular techniques!

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6th ONU Intro Molecular Biology SymposiumFirst session

Monday, February 14, 2011

Department of Biological and
Allied Health Sciences
Mathile Center 108
February 14 & 17, 2011

The ONU Intro Molecular Biology Symposium takes place every Fall and Winter quarters, as part of the Introduction to Molecular Biology (Biol 217) class. Speakers are students registered in the class, who throughout the quarter have written a review paper on molecular biology-related topics.

Click on pic for a full size image

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LectureChapter 11 - Gene regulation at the RNA levelChapter 12 - Processing of RNA

Today we finished chapter 11 by discussing how micro RNA (miRNA) is used to block translation of mRNA, how transcriptional attenuation (premature termination of transcription) is prevented by the action of RNA binding proteins, and how riboswitches can act to prematurely terminate transcription or inhibit translation.

In chapter 12, we talked about the kinds of RNA that can be found in prokaryotes and eukaryotes, and what are their relative quantities, compared to other organic materials in the cell. Then we had an overview of how different kinds of RNA (tRNA, rRNA, mRNA) are processed.

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Module 4, Lab 14 - BioinformaticsA simple bioinformatics pipeline to infer a phylogeny

A 'good' electropherogram of the RRss gene from an earthworm (Lumbricus; top),
and a 'bad' electropherogram of RRss gene of a cnidarian (Hydra; bottom)
(click pic for a full size view)
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During this lab students followed a simple bioinformatics pipeline (workflow) to process data similar those generated by them in the lab. We started by visualizing DNA sequencing electropherograms and analyzing the differences between a 'good' one (from which reliable information can be extracted) and a 'bad' one (from which no reliable information can be obtained).

Then we did a crude editing of the information contained in them and exported the data in fasta file format. Once we pooled sequence data (Ribonucleotide reductase small subunit [RRss], a nuclear gene) from 9 animals in a single fasta file we did a multiple sequence alignment using an on-line version of ClustalW available on the European Bioinformatics Institute website.

The alignment was used to generate a nexus file which can be used for performing phylogenetic analyses. We ran two, very basic analyses, one under the criterion of maximum parsimony (MP) and one under the criterion of maximum likelihood (ML), using an online version of PHYLIP, available at the Mobyle@Pasteur web portal. Students will compare the outcome of both analyses and write a short essay about the process followed to obtain the phylogenies.

The following learning outcomes should have been met:

• Introduction to the concept and the field of bioinformatics
• Introduction to the main sequence data repository in the Americas and one of the main in the world: NCBI
• Introduction to the main database in the NCBI website: GenBank
• Understanding of how to interpret an electropherogram (for DNA sequencing)
• Basic understanding of the fasta and nexus file formats
• Basic use of the BLAST algorithm
• Introduction to the concept of sequence alignment
• Basic understanding of how to perform a phylogenetic analysis

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Module 4, Lab 13 - BioinformaticsUsing the BLAST

DNA sequence alignment viewed with the multiple alignment editor Se-Al. On the far right there are insertions and/or deletions in several sequences (click pic for full-size image)

We a small and crude (quick and dirty?) overview of some of the few tools available to work in bioinformatics.

The main focus of our exercise was the use of the algorithm known as BLAST (Basic Local Alignment Search Tool), available at the NCBI webpage. After an overview of some of the main features of the site, we "BLASTed" (used BLAST) a few unknown protein (using 'protein BLAST') and nucleotide (using 'nucleotide BLAST') sequences. We found out what the sequences were and to what organisms they belonged. This is one of many possible uses of the BLAST algorithm.

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Chapter 11 - Regulation at the RNA level

Following with mechanisms of gene regulation at the RNA level, we discussed how translation can be repressed or activated by regulatory proteins and how can it be regulated via anti-sense RNA or alterations to ribosomes (we used phosphorilation of proteins in the ribosomal small subunit as an example).

We also discussed the role of RNA interference (RNAi) as a defense mechanism of eukaryotic cells against some viral infections, as a cell's mechanism for gene silencing, and as a research tool to study gene function by knocking-out genes.

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Module 4, Labs 13 and 14 - Bionformatics

The following links will provide fast access to the web pages you need to do your bionformatics exercises:

• National Center for Biotechnology Information (NCBI)
• DNA Data Bank of Japan (DDBJ)
• European Bioinformatics Institute (EBI)

• GenBank statistics
• Clustal W
• Phylogeny software

• NCBI's Coffee Break

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The animal with the most genes: A new (and unexpected) record holder

FRESH OUT OF THE PRESS!!!

Water flea, Daphnia pulex
Picture published by NPR.org
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A new complete genome has been sequenced and a new surprising fact was unveiled: The new record holder for having the most genes among animals is the very tiny water flea, Daphnia pulex (Crustacea: Branchiopoda: Cladocera). It has approximately a whooping 31,000 genes! (compared to about 23,000 in humans). Their genome, nevertheless, is not too big, which makes the surprise even bigger: It is about 200 million base pairs (compared to the 3 billon in the human genome).

This is another example of how size and morphological complexity are poor predictors of genome size or number of genes (and viceversa). Water fleas are typically no more than a few millimeters long and, to the unaware, rather unremarkable little fellows. However, they are physiologically very complex, and this seems to be the reason for which they have accumulated so many genes over evolutionary time.

Yet another surprise that the growing field of Genomics has given us.

Click here for an extended version of the news on NPR.org.

Click here to access the abstract of the original paper on Science Magazine.

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Exam 2

Stats for exam 2:

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Module 3, Lab 12 pGLO Small Scale Plasmid DNA Purification and Restriction Enzyme Digestion (RED)

pGLO plasmid and restriction map
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Today we used the bacteria we transformed with the pGLO plasmid and cloned a few weeks ago to perform a small scale plasmid DNA purification (minipreps) and isolate the pGLO plasmid again.

Then we performed a restriction enzyme digestion, RED, using the restriction enzymes EcoRI and HindIII (see restriction map).

(note: section 2 performed these procedures on Monday)

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LectureChapter 10 - Gene regulation in eukaryotesChapter 11 - Regulation at the RNA level

Today we talked about the important role that the histone remodeling complex plays in allowing proteins access to DNA in nucleosomes. We related the action of this enzyme, as well as HATs and HDs in the gene expression process as a whole.

We then discussed the role of methylation patterns in gene silencing and provided an example in genetic imprinting.

We also started a discussion on chapter 11, on how gene expression is regulated between transcription and translation (control at the mRNA level). We mentioned the several ways in which the translation of a mRNA molecule can be controlled, and then we focused in one of them: Controlling the stability and the rate of degradation of the mRNA (usually via ribonucleases a.k.a. RNases).

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Lecture, chapter 10 - Gene regulation in eukaryotes

Nucleosomes with deacetylated and acetylated histones

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After summarizing the main points of gene regulation in prokaryotes we started discussing mechanisms used by eukaryotic organisms.

We are building upon what we already know about gene regulation in prokaryotes and highlighting the added complexity found in eukaryotes. Some of the reasons for which additional or more complex mechanisms are required in eukaryotes are:

• DNA is found in a nucleus
• DNA is wrapped around histones
• Nucleosomes can be condensed in heterochromatin
• In multicellular eukaryotes different genes are expressed in different cell types or at different stages of development

We started explaining how a cell solves the problem of accessing DNA that is highly condensed (heterochromatin). Acetylation of histones, specifically of lysine residues, holds the key. We described how histone acetyl transferases (HATs), and histone deacetylases (HDs) add and remove acetyl groups from lysine residues in histones.

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Module 3, Lab 11Native and denaturing polyacrylamide gel electrophoreses (PAGEs) of GFP

Image from Bio Rad

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Today we ran 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.

We specifically ran protein samples in two ways.

• A native gel, in which proteins, in their native state, migrate at different rates depending on their size (molecular weight), 3D structure, and charge.
• A denaturing gel, in which the proteins are denatured (linearized) in the presence of a detergent such as Sodium Dodecyl Sulfate (SDS) that coats the proteins with a negative charge. The resulting denatured proteins have an overall negative charge and a similar charge to mass ratio. Since denatured proteins act like long rods instead of having a complex 3D shape, the rate at which they migrate in the gel depends only to their size (molecular weight) and not its charge or shape.
  

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.

PAGE is used for separating proteins ranging in size from 5 to 2,000 kDa due to the uniform pore size provided by the polyacrylamide gel. Agarose gels can also be used to separate proteins, but they do not have a uniform pore size, so they are optimal only for electrophoresis of proteins that are larger than 200 kDa.

We will be able to compare the results in both gels, and if GFP has any activity in either one of them (through pictures taken under UV light).

Gels were stained with Coomasie G-250 and air dried for analysis.

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(Note: Lab section 2 performed this procedure on Monday)

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Module 3, Lab 10 - Protein quantitationBradford dtermination of GFP

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 from a reddish-brownish color to blue) 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 (mostly with GFP) to estimate their concentration. The problem samples were fractions obtained from the Hydrophobic Interaction Chromatography (HIC) lab.

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.

(Note: Section 2 performed this lab on Monday evening)

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Lecture, chapter 9 - Gene regulation in prokaryotes

Today we discussed the the stages within transcription at which gene expression can be regulated, whether it is via positive or negative regulation.

We also discussed the role inducers play in causing a change in shape of activators (a.k.a. transcription factors) and repressors, as well as the role of global and specific regulators in gene expression (including the concept of regulon)

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Lecture Chapter 7 - Protein structure and functionChapter 9 - Gene regulation in prokaryotes(And... The PCR Song)

We finished chapter 7, and the third stop in our road map, on how DNA organization influences protein function. We discussed the most common structural motifs of DNA binding proteins and agents that may denature or even degrade proteins.

We also started chapter 9, on regulation of gene expression in prokaryotes. We discussed the basics of positive and negative regulation and the differences in frequency of gene regulation events at transcription, translation and in between.

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The PCR song

The highlight of the day was, with no doubt, the performance of The PCR Song by students in this class. You can see the video of the original song by clicking here, or accessing the previous entry in this blog.

The exercise, meant to reinforce the reagents and steps of PCR, ended up being delightful and enjoyable one, even leading to the suggestion to use more songs that summarize processes (a molecular biology greatest hits CD?).

I am open tu suggestions and new ideas!

(the mp3 file of the Winter 10-11 Biol 217 Ensemble is available upon request!)

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PCR song

After exam 1, which showed that several people in the class are confused on what is required to perform a PCR and on the steps of the thermal cycle, I decided to reinforce the basics of the procedure that were explained in labs 4 and 5.

Students were allowed to vote on having a quiz on Friday or learning by heart and singing the PCR song. A nearly unanimous decision favoring the latter was reached. The PCR song highlights:

• Who invented PCR
• The main reagents of PCR
• The steps of the thermal cycle
• Some important applications of PCR

Tomorrow (Friday) students will sing the song in class for the opportunity to 1) properly learn the basics of one of the most popular molecular techniques and 2) earn a few points.

The song: Students in previous quarters have found this song useful and some have even used it as a ringtone... (Warning: Cheesy!)

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Module 2Lab 6 - Gel electrophoresis of the pJet1.2+GAPC gene R.E.D.Lab 7 - Sequencing reactions of the GAPC gene

Lab 6

Today we ran the gel electrophoresis of yesterday's R.E.D. to confirm the successful ligation of the GAPC gene from Arabidopsis, which we amplified through nested PCR, with the pJet1.2 plasmid.

The results were used to determine which plasmid DNA purified samples were to be used in lab 7, when setting the sequencing reactions for the GAPC gene.

Lab 7

We added forward and reverse sequencing primers (pJET SEQ F and pJET SEQ R) to the plasmid DNA purified samples that had the GAPC gene insert 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 2-5 weeks, ready to be used in the bioinformatics labs

We will discuss 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 DNA sequencing process.

(Note: Section 2 followed these protocols on Monday)

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Module 2, lab 6 - GAPC gene cloningPlasmid DNA purification ('miniprep')

Today we used the E. coli cultures Salesha and Jess inoculated yesterday to do a small-scale plasmid DNA extraction ('miniprep') using Promega's Wizard® Plus SV Minipreps DNA Purification System. Then restriction enzyme digestions with Bgl II were set to confirm that the insert (GAPC gene) is present in the plasmid (pJet1.2).

(Section 2 did the procedure on Monday [MLK day]. Thanks to Stacy for inoculating the media the day before!)

Here's a recap of what we have done so far in the last couple of exercises:

Lab 5 - Nested PCR of the GAPC gene from Arabidopsis

Lab 6 - Cloning he GAPC gene from Arabidopsis

• Ligation of PCR amplified GAPC gene onto the pJet1.2 plasmid
• Genetic transformation of E. coli with the pJet1.2 plasmid
• Cloning of genetically transformed E. coli
• Minipreps (purification of pJet1.2 plasmid)

Next, running an agarose gel to confirm the results of the R.E.D. (lab 6) and setting DNA sequencing reactions (lab 7)

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Lecture, chapter 7 - Protein structure and function

Today we talked about the quaternary level of structure in proteins, and the nomenclature of proteins that are composed by several subunits.

We discussed a classification of proteins from a molecular biology perspective and also how DNA binding proteins can read information contained in DNA sequences without undoing the double helix.

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Lecture, chapter 7 - Protein structure and function

Today we started the final chapter on the third stop in our road map, specifically on how genetic information affects protein function.

We discussed the structure of amino acids and how they are linked into polypeptides thanks to peptide bonds (a special case of covalent bonds).

We also discussed the basics of the four levels of organization in proteins, but focused specially on the secondary and tertiary levels of structure. We discussed the importance of hydrogen bonds in maintaining alpha-helices and beta-sheets, an how these are folded into 3-D structures that are crucial for protein function.

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Module 2, lab 6 (section 1) GAPC gene cloning

Click here for info on the same procedure, performed by students in section 2.

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Module 3, lab 9 (section 1) Hydrophobic Interaction Chromatography (HIC) of GFP

Click here for info on the procedure performed by students in section 2.

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Lecture, chapter 6 - Transcription

DNA looping and interaction of general and specific transcription
factors with RNA polymerase in eukaryotic cells during transcription
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Today we finished chapter 6, on transcription.

We discussed how the RNA polymerase recognizes specific DNA sequences in the promoter of prokaryotic DNA, and how it forms a transcription bubble in order to read the template strand on the DNA and synthesize the RNA molecule. We commented how the terminator causes the formation of a hairpin in the RNA transcript that causes the RNA ploymerase to "fall off" the DNA.

We highlighted the differences in transcription between prokaryotes and eukaryotes:

• the use of three kinds of RNA polymerase (I, II, and III) for transcribing different kinds of genes
• the use of general and specific transcription factors
• the role of enhancer regions in aiding the transcription process
• the need for DNA looping and the use of a mediator complex for all the transcription factors to interact with the transcription apparatus

CLICK HERE to watch a good Youtube video on transcription, or HERE for a video from the Dolan DNA Learning Center!

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Module 3, lab 9 (section 2) Hydrophobic Interaction Chromatography (HIC) of GFP

Yesterday 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 bacterial culture 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 GFP bound to the hydrophobic beads. The high salt solution increased the hydrophobicity of GFP by further exposing its hydrophobic amino acid 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 glowed with a green color (of course).

Diagram of Hydrophobic Interaction Chromatography (HIC)

GFP molecules are represented by black triangles

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Module 2, lab 6 (section 2)GAPC gene cloning

Yesterday we started the process of cloning the GAPC gene from Arabidopsis, which we amplified via nested PCR in lab 5. The lab was divided in three main steps

• Ligation (of GAPC gene on to the pJet1.2 plasmid)
• Preparation of competent cells
• Genetic transformation of E. coli

We spent most of the lab manipulating bacteria to make them competent (i.e. get them ready to uptake extracellular naked 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, which were spread on LB agar/Amp/IPTG plates and incubated.

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Exam 1

Exam stats:

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Module 3, lab 08 (section 1)Genetic transformation of E. coli with the pGLO plasmid

We followed the same process we followed with section 2.

For more information click here.

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Module 2, lab 5 (section 1)GAPC gene nested PCRGel electrophoresis (PCR round 2) and PCR purification

Today we ran the agarose gel electrophoresis for the second round of the nested PCR of the Arabiodopsis GAPC gene (which we will clone, and eventually sequence), and also purified the reaction products, in order to eliminate leftover reagents and keep only the DNA.

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Lecture, chapter 6 - Transcription

Today we started chapter 6, on transcription, covering the basics of the process.

We discussed how to decide which are the template (sense) and coding (anti-sense) strands in DNA, the reasons for which some genes are expressed all the time (housekeeping genes) and some others are expressed only some times, and included some transcription-specific terminology.

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Module 3, Lab 08 (section 2)Genetic transformation of bacteria with the pGLO plasmid

Aequorea victoria, original source of the green fluorescent protein (GFP)

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Today we used the pGLO plasmid to genetically transform Escherichia coli.

pGLO is a plasmid that has been engineered to contain 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 regulatory sequence 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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