Last Post

This blog is defunct and will not likely be updated anymore. For anyone finding their way to this website through doing similar research, please feel free to contact me by email if you have any questions.

What will I be doing?

1) I’ll be working as a consultant to a commercial greenhouse in BC. I’m doing research to find  nutraceutical and or pharmaceutical uses for unsellable product. I signed a non-disclosure agreement so I can’t say too much about what the work is.

2) Passion project. Researching and writing about the broad aspects of global food insecurity, mostly from the perspective of how science and technology can be used to improve agriculture. This topic can be broken down to two main research areas;  the current and predicted future problems with food production and why food insecurity is an issue that needs to be addressed, and the potential ways to address these problems. The largest section will be on vertical farming.

The problems with modern agriculture

- Current environmental issues: Irrigation, pesticides, runoff, deforestation this is an exhaustive issue. I plan on only introducing the main problems and focus on specifics aspects if they can be alleviated by vertical farming or some other new technology

- Future environmental issues: Desertification, Climate change. How can climate change increase food production, both positively and negatively.

Vertical Farming

- Benefits and criticisms, current state of industry

- Technology overview and future projections: cost of LED’s, etc

- How the properties of a high density, well controlled artificially lit hydroponic environment can be used to get novel benefits over traditionally grown crops. This will be discovery and the section where maybe I can contribute some original ideas and novel arguments for vertical farms and the main reason I want to learn about all these comprehensive issues. Most of the people I’ve talked to are architects with a great love for design but little knowledge of plants or greenhouses.  Consider plant breeding – under know and optimized growing conditions plants themselves can be designed to grow optimally. Currently plants are bred so fruits look perfect on grocery shelves after a long shipment, consumers won’t buy fruit that doesn’t look good so this means selecting for traits like resistance to bruising during shipping and longevity. Those shipment traits wouldn’t be important for locally grown, quickly and predictably sold produce.

Business and logistical issues like predictable yield forecast are anouther area where a well controlled environment has advantage over traditional farms. Yields and fruit maturation times are affected in chaotic in ways by the environment, and this can cause farmers to have either too much yield with not enough buyers lined up, or less product than they promised their customers. This causes the farm to lose money directly from lost sales, as well as indirectly from missed business opportunities such as pairing with retailers to organize sales when big yields are expected. Even better than predicting when fruit maturation spikes will occurs, crops can be staggered so surpluses/shortages are evened out. Reducing or reusing food waste is as important as increasing production, even in places like India where food shortages are occurring as much as 40% of food is wasted and never makes it to store shelves.

Since vertical farming hasn’t been invented yet, many thousands of little innovations and benefits, as well as problems, are waiting to be discovered. I imagine a smartly designed vertical farm to be a very intricate and sophisticated thing

Genetic Engineering

- Benefits, highlights, how GMOs are made, and current projects

- Is there any reason to be anti-GMO?

Lab grown meat

- Also called synthetic meat, artificially meat, and in-vitro meat. The technology is far away. By far the worst aspects of food production are found in livestock. What is it?

Economics and Business of Agriculture
- What factors influence food prices? Is there such a thing as Peak Wheat?
- What’s the impact of large corporations like Monsanto?
- The role of government and subsitities
- Long term societal aspects of a change in agriculture, will farmlands be reclaimed as more people move to cities where everything is high density?
Although I’m planning on focusing on those issues listed above I want to take a holistic look at food security. While there’s no immediate danger of food scarcity and few reading this are likely to have ever thought as food as something limiting imagine what your world would be if suddenly that was no longer true and for any number of reasons food became scarce. How would panic spread, how would you and your friends react and behave in such a situation, what measures governments would have to enact on their populace, what would desperate countries have to turn to. Looking to history numerous great and civilized societies collapsed or suffered greatly due to food shortages. In our modern world are those days of truly past us forever?

So far I’ve only mentioned how issues of food security could affect myself and people like myself, people for who food security has never been an issue. Food scarcity for us is far less likely than a steady increase in food prices until the market makes projects like vertical farms economically viable. Millions are starving and malnourished, in many parts of world, for them food insecurity is a very real problem. Malnutrition is preventable, without proper nutrition people stand no chance to live their lives to the fullest and reaching their potential and simple supplements of  Iodine and vitamin A would do great benefits. Along these lines there are many projects for improving nutrition in food insecure places, like golden rice and perennial rice.

3) I’ll be going to SE Asia sometime in late September. I’d like to go for a long time, as long as money allows, but I’m not sure if I can be out of the country for my Remicade transfusions.

 

Is Competence a candidate system to study quantum biology?

My time in the Redfield lab is coming to an end. I’ve been thinking about a paper on a peculiar discovery about DNA  for the last couple of weeks, and how it might be important for the field I was in. Before I get into the actual paper I’ll discuss what quantum biology is. This post is very exploratory, I was trying to learn all this as I go through the literature. The technical concepts go far beyond my layman understanding and there are many, many highly fringe ideas(including Nobel prize winners publishing terrible papers about DNA teleportation). It was my hope that as I was doing the research for this post some coherent idea or structure would form, sometype of “aha!” moment would come if I looked hard enough but nothing came together. I did learn a lot and I’m glad that I did a fairly comprehensive search on the idea, however in retrospect this topic was much to broad and speculative to have attempted. Perhaps in a year or two it would be fruitful to look again as the science matures. I found very little solid evidence of quantum effects in DNA, got confused often and conclude that most of the ideas of extra quantum information being stored in DNA and even concepts I thought were quite reasonable like DNA electrical conductance recruiting repair proteins to be largely unfounded. I did come up with an experimental idea, however it isn’t great and likely is not worth pursuing.

Quantum Biology

Several biological process have been recently been found to exploit quantum effects beyond basic chemistry. These biological process go far beyond the inherent  and ‘trivial’ quantum nature of chemicals that allow for essential properties such as the conversion of chemical energy. These are the large scale manipulations of spooky effects to gain new abilities and functions.

The classic example of quantum biology is hydrogen tunelling. The proton coupled electron transport reaction catalyzed by soybean lipoxygenase  transfers hydrogen from one molecule to anouther – without ever having to get overcome the energy barrier needed (ref 1). The movement of hydrogen can not be explained by classical physics as the proton appears to bypass the path ti would need to take. Olfaction likewise is hypothesized to be conducted by electron tunneling.

The quantum effects I’m going to focus on in depth for the following two paragraphs are entanglement in magnetoception and coherence in photosynthesis. Entanglement is when two objects share the same existence and fate despite being in two entirely separate places. Coherence is the famous one from Young’s double slit experiment, the ability of an object to be both a wave and a point particle at the same time allowing travel through all possible routes until the wave function collapses, and maybe the single most alien thing to the way we are accustomed to things working. Coherence is comprehensible to understand, but it frustrates the brain in a funny way to think about what is actually happening.

Migratory birds have long been to known to have the ability to sense magnetic fields. Early theories to explain this ability were that birds have magnetite crystals in their heads that they use as a compass. Birds do have magnetite crystals in their heads, and other organisms such as magnetotactic bacteria do use magnetite to sense magnetic fields so it was natural to assume this was the true mechanism. However two arguments have been made against this theory. Humans also have magnetite and don’t sense magnetic fields, and more importantly migratory birds ability to sense magnetic fields is completely light dependent. In the dark they lose their magnetoception. The prevailing theory is that a light sensing molecule in the photorecptor cells of these animals called a cryptochrome is responsible. When blue light hits the cryptochrome it  produces two radicals(chemicals that have single unpaired electrons) whose electrons are entangled – sharing the same existence. The crypotochrome enters a signalling state that can only be reverted if the entangled electron spins are in one particular configuration(anti-parallel). External magnetic fields can effect the probability of the entangled pair being  anti-parallel, therefor biasing how long a cryptochrome is in a signalling state, and in this way birds have a physiological sensor of magnetic fields. A great resource for information on bird magnetoception here. Magnetoception has been seen in many other diverse organisms such as fruit flies and Arabidopsis. Unfortunately, not in humans

The second ability I investigated was photosynthesis, which is a marvelous process – converting radiation of a specific into usable chemical energy and doing so well at an incredible rate of speed and with enough efficiency to make solar panel researchers envious. I like how Philip Ball put the process in his recent article, “Photons hitting an antenna molecule will kick up ripples of energized electrons — excitons — like a rock splashing water from a puddle  and move through reaction centers to deliver charge”. These excitons are coherent, the electron travels as wave through multiple paths, finds the shortest route possible and then instantaneously appears in a reaction center. Only four years ago the coherence hypothesis was confirmed, albeit at cryogenic temperatures, and in 2010 it was shown at room temperature. Computer simulations have addressed the long standing question of how coherence can last such a long time in a cellular environment, showing that environmental noise can substantial increase coherence(2)

DNA

So that’s some of the cool stuff coming out of quantum biology. It’s interesting but not very relevant. It’s also incredibly unfamiliar territory for biologists. Now for why I’m interested in it, is there anything quantum about DNA?

I struggled heavily with this literature,  trying to find what quantum effects may be present in DNA. Unlike the previous sections, there are no seminal papers, and almost every good paper approaches it from a heavy physics perspective rather than a biology perspective. What I can find is mostly in obscure journals and the theory and methods are alien to me. One of more substantial things I found was that DNA may have a quantum quirk called “Phonons”(5). Phonons aren’t particles themselves, but describe the collective excitement of electron clounds oscillating uniformly at the same frequency. A recent paper makes the claim that phonons make a large contribution to DNA structure, saying that there is missing energy required to keep a double helix together(6). I do not know what to make of this paper, it provides no evidence for it’s claims and a lot of cmments about it are skeptical, I really can’t tell. The interesting implication with phonons is that they can be informational, a base mismatch could alter the signal and be detect far along the strand. While I was studying this I also read papers the electrical conductiveness of DNA, which also has the potential to be informational as the resistance and nature of conductance can change, for instance studies have shown mismatches and abasic strands have reduced conductivity(4).

Now for the paper that inspired this investigation, “Spin Selectivity in Electron Transmission Through Self-Assembled Monolayers of Double-Stranded DNA”. The authors put down a layer of 4 different sized dsDNA sequences(26bp to 76bp long) on a bare gold substrate at room temperature. Linear polarized laser radiation hit excited electrons and caused them to be ejected from gold plate. Electrons that have spin antiparralell to their velocity were preferentially transmitted through the chiral structure of DNA and detected by a mott polarimeter. With the 76bp DNA 4 out of every 5 electrons that filtered through the DNA were antiparralel. Electrons that were not transmitted are captured by the DNA and eventually tunneled back to the grounded gold substrate. DNA is a chiral molcule, both in sequence and in double helix structure so it naturally generates a magnetic field when a charge passes through it however the authors point out that this effect isn’t thought to be enough to cause such specificity.  When the samples where damaged by UV radiation spin specificity was lost.

What is the significance of this finding? Potentially nothing. The effect is large and suprising, but so was the spin filtering effect of graphene and that doesn’t mean anything. And just because DNA has this property when aligned tightly in a physics lab doesn’t mean it has it in vivo. What initially intrigued me about this effect is that this technology, spintronics is used for data storage. Computer hardrives and solid state devices use electron spin selectivity as a way to store information. Electron spin specificity can be informational, and is also a property of lifes information storage system? Seemed like something worthwhile to investigate. However there is currently no evidence that electron spin is biologically important to DNA’s function, and this result was so surprising I don’t think anyone predicted it.

How do proteins find their DNA targets?

One of the reasons I started thinking about this is because proteins find their DNA targets exceptionally well(3). How DNA binding proteins find their cognate target sequence, and how DNA repair proteins effectively search the genome for mismatches and bad bases is intensively studied and new methods such as quantum dots allow the visualization of individual proteins, however the exact mechanisms are often still not well understood. A 2010 paper beautifully describes nucleotide excision repair proteins in E.coli scanning, hopping, and pausing along DNA to effecitively search for DNA damage. But what causes them to act in disparate ways, what made them pause? Are there local DNA signals that inform proteins where their targets are? In other words, are proteins as perceptive in DNA environments as sharks are in bloodied water?

One paper described double stranded breaks induced by heavy ion induced radiation having repair proteins recruited to them within seconds despite the breaks only being a few bp(7)! However I’ve also found some very thorough papers describing how DNA repair proteins can find their sites without invoking any type of sensing, just sliding around short distances and hopping(8). Several papers argue that repair proteins can sense disturbance in the electric field caused by double stranded breaks to help them find problem regions(a), or that DNA can mediate long range signalling of redox states by electron transport(b).  The theory that long range charge transport is well characterized, being capable of moving very quickly and over long distances(e), however the exact function is still unclear(f)

How can any of this be related to competence?

Under conditions of nutrient starvation the bacteria the lab studies become competent. A while back Rosie brought up about an interesting point about competent DNA, it’s structurally different than normal DNA. Competent DNA  has single stranded gaps and tails. Since then I’ve liked to  imagined that as cells become more and more nutrient starved their genome becomes pocked with little nucleotide gaps[hmm… now that I think about it I never considered how these gaps would distributed. I always imagined the gaps were fairly evenly distributed across the genome, but maybe they are clustered ?], and transformation with uptaken homologous DNA replaces the old junky DNA with a healthier strand. This is the DNA repair hypothesis of competence. The important thing here is that competence structurally changes DNA. So it stands to reason that if there are long range signals of DNA damage, and if under conditions where these signals are no longer clear could affect the induction of competence(unlikely) or the transformation frequency of genomic regions(maybe?).  Static electric fields are one possibility.

Weak static electric fields have been shown to have no significant effect on prokarayotic or eukaroytic cells. Static electric fields in combination with ionizing radiation cause cell death(c). The proposed mechanism is that DNA repair proteins detect electromagnetic disturbances when double strand breaks occur and are activated and recruited, but when the cells are subjected  to an exogenous electric fields they are unable to align properly(d). Maybe log phase bacteria transferred to starvation media will not be as transformable when exposed to a static electric field. I would expect uptake to not be affected, but if there is some type of electrical signaling that is lost maybe transformation and CFUs would decrease. I don’t think there’s enough evidence to justify an exploration of this idea, especially since it would be difficult to control and even if there was an effect it would likely be hard to prove the mechanism.

One thing to keep in mind is that GC rich areas are more conductive than AT regions. Many bacterial genomes, especially their coding regions, are GC rich leading to speculation this is to increase molecular signaling and repair mechanisms. Interestingly Haemophilus influenzae, the labs model organism is  AT rich.

Maybe I could come up with some type of way to test phonons or electron spin(magnetic field?) but neither seem to have any potential, and I need to move on to other things

references:

1) http://www.chem.uiowa.edu/faculty/kohen/group/Journal/C&B99K&K.pdf

2) http://jcp.aip.org/resource/1/jcpsa6/v129/i17/p174106_s1
3) http://www.nature.com/nsmb/journal/v15/n8/full/nsmb.1441.html
4) http://pubs.acs.org/doi/abs/10.1021/jp100264v
5) http://pre.aps.org/pdf/PRE/v76/i3/e031917
6) http://arxiv.org/PS_cache/arxiv/pdf/1006/1006.4053v2.pdf
7) http://www.ncbi.nlm.nih.gov/pubmed/15913400
a) http://informahealthcare.com/doi/full/10.1080/09553000902781121
b) http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2902267/#R5
d) http://www.sciencedirect.com/science/article/pii/S1568786410000054#fig1
e) http://www.scopus.com/record/display.url?eid=2-s2.0-0033079847&origin=inward&txGid=LawXniJQNRxLYzdJBHFlz5A%3a2
f) http://www.sciencedirect.com/science/article/pii/S0027510799001955
c) http://www.sciencedirect.com/science?_ob=RedirectURL&_method=outwardLink&_partnerName=3&_origin=article&_zone=art_page&_targetURL=http%3A%2F%2Fdx.doi.org%2F10.1080%252F09553000902781121&_acct=C000050484&_version=1&_userid=1022551&md5=63aa0263135ec0f07ffda445bab5f909
8) http://www.ncbi.nlm.nih.gov/pubmed/19290859

Digesting clones

Today I did a plasmid prep of some overnight E. coli transformants and set up a digestion. These clones were found to have the rec1 insert by PCR, I’m digesting with Hind III to linerize and mlul I to check directionality. Hind III is a relatively slow acting restriction enzyme (10,000 units needed to digest 1 ug of DNA in 1 hour) so I’m leaving them in the 37 degree incubator overnight. Tommorow I’ll run a gel and if all goes well I have a 87.5% chance of finding clones with the insert in both directions

 

Edit: My mistake! 10 000 units is the total amount of units you get when you purchase a tube from NEB. 1 unit is needed to digest 1 ug of DNA in 1 hour. I did not need to leave the digest overnight

Haemophilus transformation check results

 

 

 

 

Last week I did some transformation assays to double check some mutants that we have conflicting data for. I did three different sets of experiments. Notable findings: LigA had a reduced transformation frequency when we expected normal transformation, and for RR3116 I expected a ten fold decrease in transformation but had too many colonies to count on those plates . Everything else lined up with expectations

Raw Data

K  = 9.42E-04 expected: normal

comF = 1.63E-03 expected: normal

 

K2 = 1.27E-03 expected: normal

ligA 9.03E-05 expected: normal

pilF2 7.20E-09 expected: No transformants

 

K2 7.26E-04 expected: normal

comJ 7.86E-04 expected: normal

RR3123 1.79E-03 expected: normal

RR3116 Too many to count expected: 10 fold down

comP 7.69E-09 expected: No transformants

 

 

 

 

RSS feed

I added a RSS feed to the sidebar. The feed displays the titles of the seven most recent papers on google scholar that include the terms “Haemophilus influenzae competence”. Instructions on how to make your own feed courtesy of  http://nsaunders.wordpress.com/

Todays experiments

The re-restreak plates looked good this morning so I’m running 12 test colonies through PCR to see if any of them amplify the 2815bp Rec1 gene region. I poured 2 more LB +X-gal+Ampicillin plates to use as grid plates to save the colonies I checked by PCR.

My protocol was to use a toothpick to pick up a single colony form the re-restreak plates, streak out in a grid on the grid plates, and then dip the toothpick and twirl it once in 50ul PCR tubes. I used 50ul PCR reaction mixes so the colonies are even more diluted and the various cell debris won’t interfere as much with the reaction. I kept the PCR tubes on ices, and then added a 10 minute 95 degree hot start to my PCR protocol to lyse the cells. An alternative way someone suggested to do colony PCR is to diluted the colonies in TE first and then a small amount to the PCR mixture.

My controls are a No DNA control, a Positive(map 7) control, and a Negative colony PCR control – one of my re-streaks yielded blue colonies. The blue colony negative colony PCR control should not yield any product with my Rec1 primer pair.

Tomorrow I’ll run the products on a gel. If any colonies yield a 2815bp band, I’ll go back to the grid plates and do digests to confirm and check orientation. Before I do that I need to look up what cut sites are present in both the vector and the Rec1 gene product and come up with a system to make asymmetrical cuts to determine orientation as well as product size

 

I’m wrapping up the final haemophilus transformation assay.

 

12 test

Re-re-streaking

I came in today hoping to do some PCR on my clones for the rec1 assignment, but unfortunately most of my plates had to high of a density of cells to pick out individual colonies. The point of the restreak was to be able to isolate individual clones and I had streaked out too many on cells. I poured some fresh LB+X-gal+ ampicillin plates and streaked out my ten colonies again(from the restreak plates) this time trying to take fewer cells and streaking them out over half a regular size plate instead of splitting the plates into quadrants. I also took the opportunity to restreak 8 more colonies from the original rec1 transformation plates that I had stored in the fridge, trying to cover a wider variety of colony sizes in case the rec1 plasmid had an effect on colony growth. Tomorrow I should have no problem picking out individual colonies. I’ll also be wrapping up the Haemophilus transformation assignment I’m doing for Rosie.

Previous Older Entries

Follow

Get every new post delivered to your Inbox.