Module 1, lab 1 (section 2) - RED of lambda DNAGel electrophoresis

Students completed ran an agarose gel electrophoresis of the restriction enzyme digestion performed last week. For more details check this previous blog entry.

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A bacterium that uses arsenic (and criticisms of the original paper)

Gammaproteobacteria GFAJ-1,
a bacterium capable of using arsenic as a component of its cell machinery (photo: NASA Astrobiology)

Left: Felisa Wolf-Simon, NASA astrobiology research fellow, processing
mud samples at Mono Lake. Right: Mono Lake, California (photos: NASA Astrobiology)
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Yesterday NASA made an exciting announcement in biology:

"Researchers conducting tests in the harsh environment of Mono Lake in California have discovered the first known microorganism on Earth able to thrive and reproduce using the toxic chemical arsenic. The microorganism substitutes arsenic for phosphorus in its cell components."

(From NASA Astrobiology)

This is a major finding, with important implications in the fields of astrobiology, microbiology and molecular biology, since P is one of the six elements so far believed to be essential to every life form. The Gammaproteobacteria GFAJ-1 is the first exception ever found to that rule. It was found in Mono Lake, California.

The discovery was published on the on line version of Science Magazine, and will soon be published on the regular paper edition. Pdfs of the article and support material can be accessed in my p-drive (a-cordoba. ONU students and faculty only).

In the following weeks, as we learn more about this discovery I'll bring new information into the classroom. In the meantime I want to provide links for you to start your own exploration of the topic:

• NASA announcement
• NASA Astrobiology
• National Public Radio (NPR)
• Wired Science
• CNN
• Discover Magazine
• Popular Science

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Update, March 21 2011

The news is exciting indeed, but there are detractors. Here I give them a voice (more updates will come if I find the sources)

Felisa Wolfe-Simon (of arsenic infamy) is no more convincing in person than in print
By Michael Eisen | March 18, 2011

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Update, June 01 2011

Science Magazine will publish a paper with criticisms to Wolfe-Simon et al.'s paper.  Click here to see the press release on Science Magazine News. (Link to the Science article will follow soon)

Click here for another Science Magazine News article on the criticism's to Wolf-Simon's paper.

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Lecture, chapter 3 - DNA, RNA, and proteins

Today, on chapter 3 we discussed the basic structure of nucleic acids, from the components of a nucleotide to the specialized regions in a eukaryotic chromosome. We also mentioned the central dogma of molecular biology and mentioned the several general and special cases of flow of genetic information.

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Module 1, lab 01b (section 1) Restriction enzyme digestion of lambda DNAGel electrophoresis

Today we ran the first agarose gel electrophoresis of the quarter. Students learned, or reinforced, how to load, run, take a picture of, and interpret an agarose gel.

The samples used in the gel were from the restriction enzyme digestion (RED) students set up yesterday: Lambda DNA undigested and digested with the restriction enzymes EcoRI, PstI, and HindIII. The ladder used was lambda DNA pre-digested with HindIII.

The picture below, shows the different bands of the HindIII digestion used as DNA ladder. Notice that there are 7 bands, one more than what it is specified in the lab guide. The 7th band is so faint that it is assumed to be invisible, but it was visible in most of the gels students ran. If visible, data from the 7th band should be included in the lab report.

All sizes in bp
(click on pic for full size image)
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Module 1, lab 02 (section 1) Size exclusion chromatography (SEC)

Column chromatography is a common technique used in molecular biology to purify large macromolecules, such as proteins, by separating the components of complex mixtures. A solvent (usually a buffer) and the molecules to be separated are passed through a resin of glass beads (column bed) whose specific characteristics vary depending on the type of chromatography.

Size exclusion chromatography (SEC) is a technique in which the molecules are separated by size. The glass beads in the resin have tiny pores. When the mix is applied to the column large molecules pass quickly around the beads, whereas smaller molecules enter the pores in the beads and pass through the column more slowly. The buffer and the molecules are collected in separate tubes (fractions), so that the earlier tubes get larger molecules and the later tubes get smaller molecules.

In this exercise you will separate a mix of hemoglobin (large molecule - 65,000 Daltons) and vitamin B12 (small molecule - 1,350 Daltons) using a SEC column.

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Module 1, labs 00 and 01 (section 1)Restriction enzyme digestion (RED) of lambda DNA

Micrograph and structure of a bacteriophage
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We started with a series of exercises to learn how to use a micropipette. Once students became familiar with the instrument we started with lab 1.

Lab 1 (module 1) - Restriction enzyme digestion (RED) of lambda DNA

Restriction enzymes are one of the most basic and important tools in molecular biology. They evolved in bacteria to attack and cut (cleave) foreign DNA, mostly from bacteriophages (viruses that "eat" bacteria). But hey have been isolated to be used in the lab, and are useful to cut any kind of DNA, not just viral.

Cleaving DNA is the first step in any technique that involves recombinant DNA technology. There are techniques that use special enzymes to paste (ligate) different fragments of DNA. For instance a gene can be ligated into a plasmid that can be inserted into bacteria to make many copies of it via bacterial reproduction (cloning), something we will do in a few weeks.

Today we used lambda DNA (DNA from the common lambda bacteriophage) as the substrate to be cleaved with three different restriction enzymes: EcoRI, HindIII, and PstI.

As a DNA marker, or DNA "ladder", we used a sample of lambda DNA pre-digested with HindIII.
Students will measure the distance bands in the gel migrated and will infer the size of the different bands based on such information.

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Biol 217, Winter 2010-11

Welcome to the Winter 2010-11 version of the Intro Molecular Biology (Biol 217) class.

Today we had our first official meeting, and due to several last minute registered students there has been a change in the meeting room. The class was originally scheduled to meet in Mathile 107, and indeed that's the room where we met today. But starting Friday we will meet in Meyer 128.

Today we reviewed the syllabus, explaining the grading scheme, some assignments (literature review paper and symposium presentation), and expectations in the class. We also went over the rationale of the class and how it explains the sequence of lectures that will be taught.

Reminders:

• Fall 2010 power point presentations are available on WebCT and the p-drive (under a-cordoba)
• This quarter's power point presentations will be made available as lectures are taught
• This blog can be used as a reference of the class progress; check it often, specially if you have missed class

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Module 1, labs 00 and 01 (section 2)Restriction enzyme digestion (RED) of lambda DNA

Today we had the firs lab meeting with section 2 in the class.

We went over the lab syllabus, distributed materials (lab notebooks, lab coats, and permanent markers), introduced the lab routines (where to find materials and how to behave in the lab), and spent a fair amount of time in the proper use of micropipettes.

We then performed lab 00, which allows students to practice pipetting techniques, and then we started lab 01, a restriction enzyme digestion.

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Exam 3 - Final

Stats :

Click on pic for a full size image

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5th ONU Intro Molecular Biology Symposium

Department of Biological and
Allied Health Sciences
Mathile Center 107
November 11 - 12, 2010

The ONU Intro Molecular Biology Symposium takes place every Fall and Winter quarters, when the Introduction to Molecular Biology (Biol 217) is taught. 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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Module 4, Lab 14 - Bioinformatics (Phylogeny)

When DNA samples are sent to a sequencing facility the return are files with information. The specific files that a researcher receives are called electropherograms ("recordings of the separated components of mixtures produced by electrophoresis”).

We did a basic analysis of some electropherograms of the Ribonucleotide Reductase Small Subunit (RRss) gene from animals of several phyla. We saved the information in fasta format and did a multiple sequence alignment using ClustalW. We generated a nexus file which was finally used to do crude phylogenetic analyses using the software package PHYLIP on the web.

The exercise was just an example of one of the many possible sequences of steps that can be followed to analyze genetic information. The main point was to go from electropherograms to analysis, even though the ways to analyze DNA sequence data are far too many to cover in a single lab.

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Lecture - Molecular techniques (nucleic acids)

In todays lecture we focused on techniques that are used for analyzing nucleic acids. I decided to focus mainly on DNA sequencing, given how important that such technique has become in the last decade. Topics that were discussed:

• 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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Lecture - Molecular techniques (proteins)

In this lecture we had a snapshot of what could easily be a whole course just on molecular techniques. We focused on protein analysis techniques:

• Purification (column chromatography)
• Separation (SDS PAGE and two-dimensional electrophoresis)
• Detection (western blotting)
• Predicting function (using bioinformatic tools)

We also introduced the basics of techniques focused on nucleic acids, including a discussion of the constantly decreasing cost of sequencing complete genomes.

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Module 4, Lab 13 - Links for Bioinformatics lab 1

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

• Fasta format description
• ClustalW2 (EBI)

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Module 3, Lab 12 - RED of pGLO plasmid

Today we did a restriction enzyme digestion (RED) of the plasmid DNA purifications (minipreps) we did last week. The goal was to isolate the GFP gene from the pGLO plasmid. After the cloning process that would have been a step before purifying the GFP gene sample for further study.

We used the restriction enzymes EcoRI and HindIII (individually and in combination) to reach our goal. We confirmed the results with an agarose gel electrophoresis (students in section 2 even did a "retrophoresis"... Hopefully it was a very valuable lesson)

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Lecture, chapter 11 - Gene regulation at the mRNA level

Today we covered chapter 11, on gene regulation at the mRNA level. Regulation mechanisms in the middle ground between transcriptional regulation and translational regulation.

We discussed the control of rate of degradation of mRNA, the effect of translational regulatory proteins (activators and/or repressors), regulation by anti-sense RNA, and regulation by alterations to the ribosome.

Tomorrow we will discuss RNA interference (RNAi)

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Module 3, Lab 11 - PAGEs of GFP

Image from Bio Rad

As a follow up of Thursday's lab, we stained the gels with Coomasie G-250 stain and then air dried them for analysis.

(click on pic for a full size image)

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Module 3, Lab 12 - Small Scale Plasmid DNA Purification of pGLO

In this lab students purified the pGLO plasmid following Promega's Wizard Plus SV Minipreps DNA Purification System®.

Next week we will do a restriction enzyme digestion of the plasmid DNA in order to isolate the GFP gene from the plasmid.

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

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 the 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 tertiary 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 teh results in both gels, and if GFP has any activity in either one of them (through pictures taken under UV light).

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Exam 2

Statistics for exam 2

Click on pic for full size image

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