Module 3, Lab 08 - 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 The genes in the arabinose operon (araB, araA, and araD) have been replaced by the GFP gene.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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Module 2, Lab 05 - GAPDH Nested PCR

Today we ran a gel electrophoresis to confirm the results of the second round of PCR (using the nested primers).

Ideally, we would have purified the Arabidopsis PCR product (GAPC gene; in fact, section 1 did follow the process), but all PCRs failed. WHY?

Here's what I think happened:

When I retrieved the tubes from the thermocycler they had a very small volume of liquid at the bottom of the tube. Most of the volume (pretty much all the water, not just the water you added) was condensed at the top of the tube. That means that all the reagents were desiccated and molecules couldn't interact with each other. Result: no reaction whatsoever.

Probable cause: A glitch in the thermocycler. Most thermocyclers today have heated lids, to prevent condensation of water at the top of the tube. There is evaporation, but by preventing condensation water is always being recirculated in between its liquid and gas states and there is always enough liquid water to keep the PCR going. If the lid doesn't heat up during the process, then most of the water evaporates, condensates at the top of the tube and the reaction is ruined.

I have no idea of why the lid wouldn't heat up, since it is an automatic process every time you run a program.

Solution: I am running the nested PCRs again. The first round is in the thermocycler as I type and I triple-checked to make sure the lid was hot. I will run the second round and a gel to make sure that we have product (GAPC gene). If the reaction works, in week 5, before the ligation and transformation exercise, you will have to purify the PCR product before proceeding.

Lesson: Learn how to deal with frustration. In molecular biology many things can go wrong when following a protocol and you must keep on going. If you ever become part of a research lab you will find out, first hand, that nothing is as perfect as it looks in the published literature. Today you had a little taste of it.

We must shake it off and do it again. In this case I have to do it again (but if there are any volunteers for setting up the second round of PCRs, let me know)

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Results of make up PCRs

The following gels show the results of the nested PCR (click on the pic to see a full size image):

Lanes 1 and 2 on both gels are the positive and negative controls, of the first round PCR on the left gel and of the nested PCR (2nd round) on the right. The box on the left gel shows a faint band, which resulted from leaking when I was loading the positive control in the adjacent well.

Lanes 3 and 4 on the left gel show products of the first round of PCR, and all other lanes in both gels show products of the second round of PCR.

I have saved the products of the nested PCRs for you to purify this week and go on with lab 6.

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Exam 1

Statistics of the exam:

(click on pic for full size image)

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Lecture, chapter 6 - Transcription

Today we finished the chapter on transcription.

We discussed the process in which RNA polymerase binds to the promoter in prokaryotes, generates de RNA transcript, and reaches the terminator to end transcription (Rho-independent termination and Rho-dependent termination).

We then discussed eukaryotic transcription. The promoter is more complex (it has an initiator box, a TATA box, and upstream elements), there are three different RNA polymerases that transcribe nuclear genes (mitochondrial and chloroplast genes use other polymerases), and there are proteins, called transcription factors (general and specific), that aid RNA polymerases in the transcription of genes. Some proteins may bind to enhancer regions, upstream from the promoter, to aid in the process.

Watch the video that we saw in class (embedded action disabled in Youtube)

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Module 2, Lab 05 - GAPDH Nested PCR

Thale cress, Arabidopsis thaliana

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Thursday (Round 1)

Today we started the exercise in which students will learn the basics of nested PCR. We will work with the gene that encodes one of the GAPDH isomers, GAPC, in the thale cress (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 we will run the second run, with the nested primers.

Friday (Round 2)

Today we ran the second round of PCR, using the nested primers.

In order to do so the first round primers were degraded adding an exonuclease to the amplified samples and incubating for 15 minutes at 37ºC. Then the exonuclease was denatured by incubating the samples at 80ºC for 15 minutes.

Finally, the Arabidopsis genomic DNA was diluted (1:50) and used to set up the second run of PCRs.

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Lecture, chapter 6 - Transcription

We started chapter 6 on Transcription.

We discussed general introductory issues on the process of transcription, including terminology of components and processes (e.g. template or sense strands vs. coding or anti-sense strands, hosekeeping genes, cistrons, open reading frames [ORFs], operons, and monocistronic and polycistronic DNA).

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Lecture, Chapter 5 - DNA replication

Today we finished the DNA replication chapter.

We discussed the main differences between prokaryotic and eukaryotic chromosomes, and how the ends of eukaryotic chromosomes (telomeres) are repaired by the action of telomerases (three scientists were awarded the Nobel Prize in physiology or medicine, in 2009, because of their research on telomerases).

Among the differences between prokaryotic and eukaryotic DNA replication we mentioned how in eukaryotic chromosomes have multiple replication origins, and in their DNA replication process there are two DNA polymerases and a few proteins that are not found in the prokaryotic DNA replication.

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Lecture, chapter 5 - DNA replication

Today we covered most of chapter 5, on DNA replication. We talked about the replication fork and the replisome (all the enzymes that are part of the replication fork), including the difference in between how the leading and lagging strands are synthesized.

We also mentioned how important DNA looping is for the lagging strand to be synthesized. This video illustrates such phenomenon beautifully:

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Module 1, Lab 04 - Size exclusion chromatography (SEC)

Friday, September 17, 2010

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, Lab 03 - PCR of the human PV92 locus

PV92 locus genotypes of Fall 2010 students

(click on pic to see a full-sized image)

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Thursday, September 16, 2010

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 locus, located on chromosome 16. This locus may, or may not, have an insertion of an Alu element. Alu elements are a family of short interspersed repetitive elements (SINEs) that have mobilized throughout primate genomes for the last 65 My, by retrotransposition.

In this exercise you will find out if you have the PC92 Alu insertion in one, both, or none of your chromosomes.

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 the PV92 insertion. Because the PV92 insertion 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.

So, do you have 0, 1, or 2 PV92 Alu insertions in your genome?

The following picture illustrates the possible outcomes of your PCRs:

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

What is your genotype like?

In the mean time enjoy The PCR Song! Students in previous quarters have found this song useful to remember the sequence of steps in PCR... (Warning: Cheesy!)

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Lecture, chapter 4 - Genes, Genomes, and DNALecture, chapter 5 - DNA replication

Today we finished chapter 4.

We focused on the importance of supercoiling DNA so it fits in a cell (prokaryotes) or in a nucleus of a cell (eukaryotes). We discussed the mechanisms through which prokaryotic DNA is supercoiled and how eukaryotic DNA is packed in chromosomes as chromatin (in this case the term 'supercoiling' is not really applicable, but it is a useful analogy).

Then we started chapter 5, on DNA replication and we did a quick introduction to the replication fork and the elements involved: DNA and the replisome (all the enzymes involved in the DNA replication process)

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Lecture, chapter 4 - Genes, Genomes, and DNA

Today we covered most of chapter 4.

The main topic we covered was non-coding DNA. We talked about interspersed elements (LINEs, which are moderately repetitive, and SINEs, which are highly repetitive), and tandem repeats (satellites, minisatellites [or VNTRs], and microsatellites [or STRs]). We also talked about junk and selfish DNA, palindromes, hairpins, and stems and loops.

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Module 1, Lab 01 - Phenol-chloroform DNA extraction

Today we had our first lab (what an experience!) in which students started extracting DNA from their own blood. We had the assistance of Dr. Lisa Walden and her students from ONU's Clinical Laboratory Science, who very kindly offered to do the phlebotomies necessary to obtain the samples.

The exercise took longer than expected, so we didn't finish the process, but the samples were frozen after the cell lysis step and will be ready to continue with the addition of phenol-cholorform-isoamyl alcohol (PCI) in the next lab session.

Tomorrow: Restriction Enzyme Analysis (RED) of lambda-DNA.

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Friday, September 10, 2010

Module 1, Lab 02 - Restriction Enzyme Digestion (RED) of Lambda DNA

A micrograph of multiple bacteriopages

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.

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Lecture, chapter 3 - DNA, RNA, and proteins

Today we finished chapter 3 on the basic structure and function of DNA, RNA, and proteins.

We talked about characteristics of base pairing in DNA, characteristics and functions of different kinds of RNA, the central dogma of molecular biology, and about the very basic structure of proteins.

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Lecture, chapter 3 - DNA, RNA, and proteins

Today we had our first lecture. We introduced the course and had an overview of the syllabus. Then we had an overview of the "road map" of this course, a series of major topics that are the backbone of what the course is going to be, and how each one of the chapters fit within such plan.

We staring covering chapter 3, on DNA, RNA, and proteins. We talked about the very basics structure of nucleotides and their components, how they are linked, and the rationale behind their nomenclature. We finished the lecture talking about the double helix structure of DNA.

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Welcome to the Introduction to Molecular Biology class, Fall 2010-11

Welcome to the intro Molecular Biology class (Biol 217) of the Fall quarter 2010-11!

In this blog you will find updates of our progress in the class, both in lecture and in the lab. You can use it as a topic guide for studying for exams and make comments when ever you see it fit.

The blog will also be used as a means of posting information relevant for the class, such as external links, occasional changes in scheduling, and general announcements.

If you have any ideas to improve the blog or about information that you would like to see posted, please send me an e-mail. Cheers and good luck this quarter!

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IV ONU Intro Molecular Biology Symposium

Department of Biological and

Allied Health Sciences

Matile Center 138

February 17 - 18, 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.

In this fourth edition we had two guest speakers, who took the class in the past and presented at the symposium in earlier editions.

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Wednesday, February 17

GUEST SPEAKERS

8:00-8:30	Analysis of branching in the lycophyte genus Selaginella Eric R. Schultz
8:30-9:00	Engineering increased oil production in non-seed tissues of rutabaga (Brassica napobrassica) Alan Bowsher

SYMPOSIUM TALKS

9:00 - 9:15	Prion diseases Melissa Straub
9:15 - 9:30	DNA and its forensic use in cold cases Rachel Butvin

Thursday, February 18

8:00 - 8:15	Chimerism and its consequences on the Innocence Project Jake Lewis
8:15 - 8:30	Nanoparticles: Novel approach for the battle against cancer Will Proctor
8:30 - 8:45	Gene Therapy: A potential Cure for Cancer Brooke Fleming
8:45 - 9:00	From RFLPs to STRs: The Historical Journey of DNA profiling in Forensic Science Katelyn Avendt
9:00 - 9:15	Genetic Basis for Homosexuality in Males Sonia Dhaliwal
9:15 - 9:30	The use of CODIS in DNA Profiling, and its future prospects Lindsey Pruneski
9:30 - 9:45	Parkinson's Disease and Gene Therapy Shannon Bruewer

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Lecture - Molecular techniques

Two-dimensional polyacrylamide gel electrophoresis from E. coli protein extracts
(From Alberts et al. 2008. Molecular Biology of the Cell, 5th edition. © Garland Science)

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Today we started discussing the topic of molecular techniques. There is no single chapter associated in the textbook. The material is a summary taken from miscellaneous sources.

We covered the basics on techniques on protein analysis. We focused on column chromatography (HIC, SEC, Ion-exchange, and affinity), two-dimensional gel electrophoresis and western blotting.

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Lecture, chapter 12 - RNA processing

Today we finished chapter 12, on RNA processing.

We discussed the process of intron splicing, in which snRNPs ("snurps") play an important role, the different mechanisms of alternative splicing (promoter selection, tail site selection, exon cassette selection, trans-splicing), base modification (methylation and pseudo-urydilation), RNA editing, export of RNA to the cytoplasm, and mRNA degradation (in prokaryotes and eukaryotes).

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Lab 14a - Bionformatics

Screenshot of a typical BLAST output
(click on pic for a full size image)
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Today we had an extremely shallow introduction to the universe of bioinformatics. We had an overview of the main genetic information repositories in the U.S., Europe, and Japan (see previous post), with emphasis on the NCBI website, specially on its main database, GenBank, and one of its main tools, BLAST.

The goal for this lab is for students to get acquainted with BLAST, by "blasting" nucleic acid and protein sequences (the verb 'to blast' makes reference to using the BLAST feature in the NCBI website).

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