undergrads3

I am hoping that she will catch on to genomic DNA preps and PCR rather quickly. Because I have an upcoming project that is going to require an extreme amount of both of these techniques. After she feels comfortable with genotyping plants, we will move on to more advanced tasks such as protein analysis, gene expression analysis, and eventually molecular cloning.  I have witnessed and been told that it takes a year or more for an individual to become competent in a molecular biology lab – that is, they do not need someone to be watching their every move, they can undertake real responsibilities pertaining to the research being performed in the lab, and most importantly, obtain the ability to think independently at the level required to work in a cutting-edge molecular biology lab. I have high hopes for this undergrad, as she attended one of the most prestigious science high schools in Oklahoma. All of my explanations seem to be sinking in with ease, however, I shouldn’t speak so soon – we will see how this PCR looks…

undergrads 2

We are performing PCR to identify if 35S:NF-YB12:YFP:HA was ever inserter into the genome. We will be amplifying the region using a 35S forward primer and a gene specific reverse primer. This should tell us if the transgene made it into the genome. We could also amplify another region, using a YFP reverse and gene specific forward primer combination. We could amplify another region, using the primer combination of gene specific forward and HA reverse. One final combination, which would give you the answer, but would be a sloppy approach is using the two primer extremities of the construct, 35S forward and HA reverse. This requires doing a little math for confirmation. This requires looking at the sequence of the construct, counting nucleotides for each portion of the construct, and adding this together to get a collective base count of the construct. The amplified band should be exactly that size.

undergrads

I am currently working with a freshman undergraduate student who wants to graduate with a degree in pharmacy. Hopefully exposure to a molecular biology lab will steer her in the correct direction. Yesterday I walked her through the workings of a plant genomic DNA prep. It looks like the DNA we extracted is going to work. We will find out the answer today. We are testing whether or not the prep worked by PCR. She will be amplifying a region of DNA where I suspect there to be a transgene. I am asking her to help me with identifying the presence of 35S:NF-YB12:YFP:HA. I am a bit skeptical of this line because I have performed protein analysis and had odd results. Most of the proteins I am trying to detect are ~25 kDa, with a 27 kDa YFP marker. This should some to ~50 kDa. I am worried because I am getting bands on western blots that are only half that size. This tells me one of two things: either the protein of interest is being cleaved out, and I am detecting only YFP:HA, or the other scenario, which is that I have no idea. 

the dark side of translational fusions

Inhibitory effects of protein function are usually associated with mutations within the coding region of a gene. Inhibition can also come from additions of elements to a protein. I was reading a paper recently that showed that the C-terminal addition of GREEN FLUORESCENT PROTEIN (GFP) to a gene construction actually affects the ability of the protein to perform, in vivo. In order for plants to flower, a small protein (~25 kDa), called FLOWERING LOCUS T (FT) must be transported out of the leaves, through the petiole, loaded into the phloem, up through the plants vasculature, where it eventually reaches the shoot apical meristem to induce flowering. The small nature of this protein, accompanied with the long distance it must travel to accomplish one of its jobs, can be negatively affected by the addition of GFP, used to detect sub-cellular localization.  The translational fusion of GFP to FT decreases the efficiency of FT to drive flowering in an ft mutant background. This is supported by flowering time assays in Arabidopsis.

PAGE

Today I looked at protein expression of a flowering time experiment. I evaluated protein expression by western blotting. This is a fairly straight-forward concept that allows you to see relative amounts of protein. I began by extracting total protein from plants that I was interested in evaluating protein expression/accumulation. This process starts by clipping a single leaf from a plant and grinding the leaf in a tube with an appropriate amount of extraction buffer. The extraction buffer allows the protein to go into solution with a cold centrifugation step. After centrifugation, everything except total protein is pelleted to the bottom of the tube. I then remove this top portion, called the supernatant. This protein rich extract is transferred to a tube containing loading dye, and heated at 95 degrees for 5 minutes. This heating step denatures the protein, or linearizes it, in other words. We are now ready to load our proteins into a poly-acrylamide gel that utilizes charge to push the proteins through the gel. The different proteins are separated by size, larger proteins remain higher up on the gel, smaller ones drift further down.

Watson and Crack, fleshed out.

We wish to suggest a structure for the salt of deoxyribose nucleic acid (DNA). This structure has novel features that are of considerable biological interest in that it suggests a copying mechanism for genetic material.

            Several structures for DNA have been suggested, but all are unsatisfactory in our opinion. Our structure uses molecular architecture similar to Fraser’s model, proposing that phosphates occupy the outer extremities of the chain, with the bases making up the central region. We propose that DNA consist of two helical chains each coiled around a central axis. Both chains follow right-handed helices, however, their orientations are anti-parallel. Each chain is composed of a phosphate-deoxyribose backbone, with 3’-5’ linkages.

            Four molecules make up the structure of DNA, two pyprimidines, cytosine and thymine; and two purines, adenine and guanine. For bonding to occur, specific purines must join by hydrogen bonding with specific pyrimidines: adenine to thymine and cytosine to guanine, respectively. Bonding specificity is consequence of the molecules taking keto-configuaration. Our model is experimentally supported by reports claiming that ratios of adenine to thymine, and cytosine and guanine are always close to unity in DNA.

            This model is biologically significant because it proposes that phosphates are readily available to be accessed by the cell’s excess of cations. Our model also assumes that DNA is an open, hydrophilic molecule, thus, highly water-soluble. Alternatively, at lower water content, DNA’s thermodynamic favorability is predicted to tilt its bases so that the structure can become more compact.

            

flowering

Light has a strong effect on the stability of proteins that regulate many physiological and developmental processes in animals and plants alike. Circadian rhythms depend on rapid accumulation and degradation of protein products within a 24-hour time window. Light to plants is more than a source of energy to generate sugars; it is an external timing apparatus (for most plants) that allows for the plant to reproduce when environmental conditions are favorable for offspring development. For floral initiation in Arabidopsis to take place, 16 hours of light per 24-hour period, is preferred. Light strikes the photoreceptors, PHYA, PHYB, and CRY2, which then entrain the circadian clock to trigger the expression of CONSTANS (CO). CO is a transcriptional regulator that is expressed (under appropriate conditions) in the leaf vasculature in most long day (16 hours of light, 8 hours of darkness).  CO expression follows an oscillatory trend throughout a 24-hour period, as the protein accumulates in light and is rapidly degraded in the dark. With the help of the transcriptional regulators that the Holt Lab studies (NF-Y), CO can bind to the promoter of a gene called FLOWERING LOCUS-T (FT). FT acts as the central hub for many flowering pathways. After FT has been activated, the protein product (produced in the leaves) travels against a natural gradient, through the phloem companion cells, where it eventually reaches the shoot apical meristem to turn on another group of transcription factors that induce flowering. 

flowering

Plants, like animals, respond to environmental factors. Light seems to be the most influential external factor on the planet (omitting the anomaly of hydrothermal vent life of oceanic trenches). Light is key to plant life for synthesis of usable sugars, as well as reproduction. Floral initiation, in Arabidopsis can occur through a number of pathways, given that Arabidopsis is a facultative, long day plant. This means that is favorable for Arabidopsis to flower when day lengths stretch into 16 hours of light and 8 hours of darkness, per 24-hour period. Alternative floral initiation pathways can be triggered if this light regiment is not met. An alternate pathway group is influenced by temperature acting as the environmental factor. Vernalization, autonomous, and ambient floral initiation pathways are triggered by variable temperature exposure. A pathway independent of environmental factors would be the Gibberellic Acid pathway. This is a plant hormone that is known to positively regulate seed germination, floral initiation, and many other physiological and developmental processes. 

RNA IV

Poly-A mRNA purification starts by linearizing your total RNA. This is a necessary step because it is thermodynamically favorable for RNA to hybridize back onto itself, creating secondary structures called “hairpin loops”. We must linearize our RNA to remove secondary structures, which ultimately exposes the polyadenylated tail of the mRNA transcripts. After this step, we add our linearized transcripts into a magnetic column containing single thymine molecules (adenines bind to thymines) bound to single Iron atoms. Through binding affinity, we are able to grab individual mRNA transcripts, while allowing any molecules lacking polyadentylated tails to flow through the column. The mRNAs are stuck to the wall of the tube, bound by the iron atom/thymine matrix, which in turn is magnetically attracted to the wall of the tube that is placed in a slot of a device possessing a strong charge, opposite that of iron. We are now ready to elute our purified mRNA. I choose to do this with RNAse free water. I once again determine quality and quantity of my extract by spectrophotometry. 

RNA III

After total RNA isolation and a successful DNA digestion, we need to purify mRNA from our total RNA extract. Total RNA consist of somewhere between 1-5% mRNA (organism dependent) and the other ~95% contains ribosomal RNAs, TRNAs and other small RNAs. Eukaryotic systems have an advantage over prokaryotic systems due to a specific biochemical advantage at the 3’ end of each mRNA transcript. This advantage is a polyadenylation event, or a 15-30 base pair adenine rich tail (consistent adenine repeat) at the end of each transcript. This is advantageous because we can design a “handle” that will, without fail, hybridize to our polyadenylated transcript. In order to isolate an efficient amount of mRNA for RNA-seq (~500ng) you need to start with a large amount of total RNA (remember that only 5% of total RNA are mRNAs), something around 3mg of total should be efficient. Quality of mRNA input into an RNA-seq experiment is extremely important. If care is not taken in the cleanup step, you might as well start over.

RNA II

We start the protocol by freezing our samples immediately in liquid nitrogen to solidify the transcript that we are going to capture, this also disrupt any RNAse activity that could potentially degrade our transcript. We then grind our frozen tissue a in a frozen mortar with an RNA extraction/lysis buffer. We add this slurry of tissue/buffer into a homogenization column and centrifuge to filter through the RNA rich liquid we are after. Next comes the precipitation of RNA. This is facilitated by absolute ethanol. We add the ethanol/buffer+RNA mixture to a separate column that has high binding affinity to RNA. The RNA precipitates after a short spin and binds to the column matrix. We then add several wash buffers to “clean up” our sample that is soon to be eluted.  The second wash buffer contains ethanol that has been diluted to 70%. This allows for both ethanol and water soluble contaminants to be discarded. While isolating RNA, there is always genomic DNA contamination. This occurs due to shared biochemical properties between the two molecules. I prefer to do an “on column” RNAse free DNAse treatment that supposedly degrades 99.99% of contaminating genomic DNA. After this step, we are ready to elute our clean, total RNA. I use a spectrophotometer to determine the quality and quantity of my extraction.

RNA I

The overarching goal of my Transcriptomics (RNA sequencing) class is to provide an outline, or protocol to send samples for RNA-seq that would work for anyone working in our system. This is a great idea because we are getting a wide range of protocols covering a wide range of systems. All eukaryotic systems are virtually identical, however, the prokaryotic crowd encompasses aerobic and anaerobic organisms with radically variable needs.

The starting place for an RNA-seq protocol begins with total RNA isolation. This process differs between plants and animals due to the presence of a cell wall in plants. Plant RNA isolation is a fairly simple process, if using a kit. Before one can begin to isolate this fragile nucleic acid, certain procedures must be taken and potential problems must be addressed. First, RNA is single stranded, thus lacking the hydrogen-bonding matrix that DNA, a double stranded nucleic acid contains. This property makes RNA very fragile. Second, our skin, breath, and hair are riddled with proteins called RNASes. These are proteins that target RNA and chop the transcripts into untranscribable messages, thus destroying the polynucleotide. While isolating RNA, one must not take these two considerations lightly. We ALWAYS use gloves and RNAse/DNAse free materials including water, tubes, isolation columns, and all solutions used throughout the isolation process, as well as any downstream single stranded nucleic acid application.

Natural Variation

Evolution is a driving process that takes in information as an input (typically genetically inheritable information) and spits out deviants or variants of this genetic template. Natural variation among species is the results of continual evolution. Natural variation occurs at all hierarchical levels from kingdoms to families to genus to species. For example, the organism used as a model species in the plant biology world is Arabidopsis. Since the early 21^st century the Arabidopsis community has undertaken the laborious task known as the one thousand and one genome project. In other words, they are sequencing the genome of every Arabidopsis ecotype. We use three of these fully sequenced ecotypes in our lab for various reasons – simply, some are better for certain assays than others. This is an example of natural variation at a large, topical level. Let us consider natural variation at the molecular level. Just as whole genomes differ slightly from their common ancestor, the family of proteins I work on has a large region of homology (~70%) with one another. The landscape at the sequence level, protein and DNA alike follow a trend of each other. It is unknown whether or not these variations account for evolutionarily induced new functions of the proteins themselves. However, the answers are being slowly uncovered. 

untitled

Scientific writing can go one of two ways, interpretable or the opposite. I feel the “middle ground” in scientific writing is under or less represented than writing of other backgrounds – you either get the message or you don’t. It is difficult for a research scientist to step out of a world filled with acronyms and lab jargon to get a relatively (in most cases) simple idea across to the general public. This might be the most challenging task that scientist face today. Whether it is information the researcher is trying to convey through a talk or presentation, write-up or report of some fashion, we are challenged by the task of relaying information. It is possible that the ideas and information come so clearly to researchers in this field because we live in a world that is riddled with simple and complex variants of the central dogma. The same could be said for any professional that finds his or her work inherently and naturally understandable because they are engulfed in the world. Relaying complex information’s requires one to step back, and ask what is the take home message here, and how can I get to that point as clearly and concisely as possible? I have learned that making analogies to my research to be one of the more effective methods of describing my research to people outside of the scientific community. 

Boulder, CO cont.

Next, we went to Pearl street, this is the awesome street with the wide array of food choices, awesome little shops, street musicians, basically the street that every perfect college town has laying behind its campus. Massachusetts street in Lawrence, Kansas and State Street in Madison, Wisconsin are examples. There was a cool restaurant tucked away in a pedestrian zone about half a mile into our walk. We drank our beers on the rooftop patio overlooking the mountains. We spent four hours I'm boulder and in that short amount of time I was convinced that it is bar far one of my favorite college towns. The weather was a beautiful 70 degrees and sunny, I guess they experience an earlier exposure to fall. Driving back into Oklahoma, I had noticed the temperature was roughly identical to what I had become used to while spending a weekend in CO. This surprised and excited me at the same time – I had left OK when temperatures had remained constant for months above 100 degrees. There is hope on the horizon.  

Boulder, CO

The drive into boulder from Denver was about 30 minute trek into canyon surrounded by towering peaks. We had initially planned to meet up with one of our friends from Norman that recently moved to boulder to peruse a PhD in chemistry. After meeting our friend Jesse, We hopped in the car and headed to the CU campus. I had heard people criticize Colorado for not giving the best college degrees, and that it's only a party school. I can see how this might be true, there is just so much to do, and every single day the weather is perfect- quite a change from what I am used to here in Norman. Everything was lush green, alive, and not 900 degrees. Just a glance to the left on a hill and enormous peaks reveal themselves. We walked from the library to the chemistry building, passing over two streams, and a ten-foot sundial in the process.