Module 3, Lab 08 - Genetic transformation of E. coli with the pGLO plasmid

Jen was kind enough to prepare new LB agar plates and redo the transformation of E. coli with the pGLO plasmid. Then she plated the transformed bacteria on the plates she prepared, some of which had ampicillin, arabinose, or both.

[Fantastic] Results are shown in the pictures below:

(entry in progress)

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Lecture, chapter 11 - RNA processing

tRNA processing

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Today we started chapter 11, on RNA processing, the fifth "stop" in our roadmap.

We mentioned the most basic processes through which RNA is modified (base modification, cleavage, and splicing) and described the more complex processes that are specific to eukaryotic RNA (5' capping and 3' polyadenylation).

We discussed in more detail the steps through which introns are spliced out the pre-mRNA molecule, and introduced the concept of alternative splicing.

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Lecture, chapter 10 - Gene regulation in eukaryotes

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

We highlighted the differences in between the regulatory processes in prokaryotes and the more complex processes in eukaryotes, mainly the facts that in eukaryotes many transcription factors are needed to aid transcription and that DNA is many times condensed in heterochromatin, and therefore unavailable for RNA polymerase and other proteins.

Among some of the processes that affect gene expression we discussed histone acetylation, a mechanism through which heterochromatin is relaxed into euchromatin, making DNA available for DNA binding proteins, and methylation, a mechanism through which genes can be repressed or silenced, in some cases because it promotes de-acetylation of histones and DNA condensation into heterochromatin.

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Module 2, Lab 06 - Ligation and transformation (GAPC gene from Arabidopsis and pJet1.2 plasmid)

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

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 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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Lecture, chapter 09 - Gene regulation in prokaryotes

Today we lectured for a little over one hour, as a replacement for the lab we could not do, because of not having transformed bacteria to "play" with.

We finished chapter 9 on gene regulation in prokaryotes. We discussed the concepts of positive and negative regulation, including the role of (specific) activators and repressors, and global regulators. We studied how the Lac operon works in E. coli, and set the stage for starting with gene regulation in eukaryotes.

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Lecture, chapter 09 - Gene regulation in prokaryotes

We started the fourth stop in our "road map" (how genes are regulated).

We introduced chapter 09, on gene regulation, by highlighting the importance of the process for both eukaryotic and prokaryotic cells. We also mentioned the different steps during the process of information transfer at which regulation can take place, transcription being the most common one.

Note: Because the results of the transformation lab were negative (E. coli genetic transformation failed) we will lecture tomorrow.

• Section 01: 8:45 am
• Section 02: 10:00 am
• (Any one who wants to attend the opposite section is welcome)

Information on how to obtain the results from the transformation lab will be posted on an update in that exercise's blog entry.

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Lecture, chapter 7 - Protein structure and function

We finished chapter 7...

We discussed the features that allow certain proteins to bind to DNA and also what of their most common structural motifs are. We closed by briefly describing what protein denaturation is, how it differs from degradation, and what are the most common denaturing agents.

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Lecture, chapter 7 - Protein structure and function

Today we covered most of chapter 7, on protein structure and function.

We discussed the properties a protein has given the characteristics amino acids provide to the whole structure and how they actually affect protein function. We also listed different protein categories according to different functions they may have.

Tomorrow we will talk about how proteins can recognize DNA sequences to bind to them.

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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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