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.

_____________________________________________________

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

_____________________________________________________

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)

____________________________________________________

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.

----------------------

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).

----------------------

Lab 14a - Bionformatics

Screenshot of a typical BLAST output
(click on pic for a full size image)
_______________________________________________

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).

----------------------

Bioinformatics links for lab 14

The following links will provide fast access to the web pages you need to do your bionformatic 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

---------------------

Lecture, chapter 12 - RNA processing

Today we started covering chapter 12, on RNA processing.

We discussed the kinds of processing that different RNA molecules undergo, in both eukaryotes and prokaryotes. We compared the kinds of RNA found in bacteria (regulatory RNA and tmRNA) with those find in eukaryotes (snRNA, snoRNA, scRNA, miRNA, siRNA...), of course, besides the "classics": tRNA, rRNA and mRNA, present in all cells.

We discussed mRNA processing more in depth than in previous chapters, and we introduced the basics of rRNA and tRNA post-transcriptional processing. When focused on mRNA we talked about 5'-capping and polyadenylation ("tailing"). On Friday, we'll talk about intron splicing.

----------------------

Exam 2

Today we had our second exam.

Statistics:

----------------------

Lab 12 - Polyacrylamide Gel Electrophoreses (PAGEs) of GFP

Today we discussed 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.

Specifically we were supposed to use it to run protein samples and in two ways.

• A native gel, in which the proteins migrate at different rates depending on their size (molecular weight), tertiary structure, and charge.
• A denaturing gel, in which the proteins are denatured with high temperature and kept denatured by SDS contained in the electrophoresis buffer, so their rate of migration through the gel depends exclusively on size.

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. Unfortunately we didn't have the reagent to stain the gels (coomasie), so we didn't actually run the gels, although we did talk about the process.

----------------------

Lab 11 - Protein quantitation

Wednesday, February 3, 2010

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 to a different color) 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 to estimate their concentration. The protein samples obtained from the Hydrophobic Interaction Chromatography (HIC) are used as problem samples.

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.

----------------------

Lecture, Chapter - Gene regulation at the RNA level

Today we covered chapter 11, on gene regulation at the RNA level.

We discussed mechanisms of gene regulation between transcription and translation:

• Rate of degradation of mRNA
• Modification of mRNA
• Control of translation by RNA binding proteins
• Anti-sense RNA
• Ribosome alteration (preferential translation of mRNAs)
• RNA interference (RNAi)
• Micro RNA (miRNA)
• Riboswitches

----------------------

Lecture, chapter 10 - Gene regulation in eukaryotes

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

We discussed the role of DNA looping and insulators in ensuring that enhancers have an effect only on associated genes.

Then we discussed how a cell deals with the fact that much of its DNA is found as heterochromatin, making it inaccessible for RNA polymerase, and how DNA must be turned into euchromatin to be able to be expressed... or how euchromatin can be turned into heterochromatin to turn genes off (histone acetylation/deacetylation, DNA methylation/demethylation).

We also introduced the concepts of gene silencing and genetic imprinting

----------------------

Lab 7b - Smal scale plasmid purification (pJet1.2 with GAPC gene insertLab 13 Small scale plasmid purification (pGLO plasmid)

Today we used the bacteria we transformed (and cloned) with the pGLO and pJet1.2 plasmids to perform small scale plasmid DNA purifications (minipreps) and isolate the plasmids.

Then we performed restriction enzyme digestions, RED, using the restriction enzymes Ec0RI and HindIII (see restriction map) for the pGLO plasmid, and BglII for the pJet1.2 plasmid.

The idea was to isolate the GFP gene in a fragment, in the case of pGLO, and to confirm the success of the ligation procedure in the case of pJet1.2.

----------------------

Lecture, chapter 9 - Gene regulation in prokaryotesLecture, chapter 10 - Gene regulation in eukaryotes

Today we finished chapter 9, on gene regulation in prokaryotes, and started chapter 10, on gene regulation in eukaryotes.

We discussed the ways in which a protein can be a promoter for some genes and at the same time a repressor for others, and the role an inducer may have in changing its tertiary structure.

We also compared the action of global vs. specific regulators and introduced the concept of regulon.

Finally, we discussed the anti-termination control mechanism for regulation of gene expression.

We talked about the reasons control of gene expression in eukaryotes is more complex than in prokaryotes (supercoiling, presence of a nuclear envelope, cell differentiation in multicellular ones, developmental reasons, etc.).

We describe how the transcription apparatus is assembled and the role the mediator complex plays in facilitating its interaction with specific transcription factors.

----------------------

Lecture, chapter 10 -Gene regulation in prokaryotes

Today we stated chapter 10, on gene regulation in prokaryotes.

We discussed the importance of having gene regulation mechanisms in place for the correct functioning of prokaryotic cells, and why it is more common to find such mechanisms acting upon transcription than other cellular processes.

We also discussed the difference between positive and negative regulation, the role activators and repressors play in such processes, and how they are affected by the presence of inducers in the environment.

The basics of the operon model of gene regulation were also covered.

----------------------

Lab 10 - Protein purification(GFP) through Hydrophobic Interaction Chromatography (HIC)

Today 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 bacteria 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 the naturally hydrophobic GFP bound to the hydrophobic beads. The high salt solution increased the hydrophobicity of GFP by further exposing its hydrophobic 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 should glow.

----------------------

Lab 07 - Ligation and transformation (GAPC gene from Arabidopsis and pJet1.2 plasmid)

Wednesday, January 20, 2010

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

The lab was divided in three main steps

• Preparation of competent cells
• Ligation
• Genetic transformation

During most of the lab students manipulated 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 that were spread on LB agar/Amp/IPTG plates and incubated.

----------------------

Lecture, chapter 7 - Protein structure and function

Today we continued with the chapter in protein structure and function.

We discussed the most common shapes found in the secondary structure of proteins, α-helices and β-sheets. Me mentioned the typical ways in which they refold to obtain their tertiary structure.

We talked how can proteins "read" DNA information without separating the strands in the double helix and the most common DNA-binding motifs found (helix-turn helix, helix-loop-helix, leucine zipper, zinc finger).

----------------------

Module 3, Lab 9 - Genetic transformation of E. coli with the pGLO plasmid

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

First lab in module 3. Today we used the pGLO plasmid to genetically transform E. coli.

pGLO is a plasmid that has been engineered to contain and express 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 activating mechanism 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.

----------------------

Lecture, chapter 6 - Transcription in eukaryotesLecture, chapter 7 - Protein structure and function

Today we finished chapter 6, on transcription, specifically in eukaryotes.

We discussed the mechanism in which general and specific transcription factors aid RNA polymerase II in initiating transcription of protein-coding genes in eukaryotes. We also discussed the role of enhancer regions as sequences recognized by specific transcription factors and the role of DNA looping to allow the interaction of transcription factors that bind to the DNA far away from the gene (or transcription unit).

We also started chapter 7 on protein structure and function. We covered the characteristics of amino acids and then we started discussing the levels of structure in polymers, specifically polypeptides.

----------------------

Exam 01

Today we had our first exam of the quarter.

Statistics:

----------------------