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 The genes in the arabinose operon (araB, araA, and araD) have been replaced by the GFP gene.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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