How long is a piece of stri…promoter?

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Trying to work out how long to make the upstream promoter region for some plasmid constructs. I’ve been using the SnapGene tool to visually stitch together DNA sequences.

How long is the promoter for a particular plant gene? 500, 1000, 2000 bp? or is it defined by particular features in the promoter? I guess for some genes the upstream region barges into other gene loci.

I tried out this web-tool, plantpromoterdb.

Seems quite useful. It displays some features of the promoter that might be considered when deciding the promoter chunk length e.g. it will show the predicted transcriptional start site, and other cis– features.

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Seems, though, that most studies take a nominal 1000 to 2000bp upstream of the translational start codon (ATG). Think it’s good to know some of the promoter ‘landscape’ though. Here’s the reference for plantpromoterdb:

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Feeling Fly

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This fly paper caught my eye. It examines how Drosophila monitors daily temperature changes via network of circadian clock regulated neurons. It seems the fly continually integrates temperature informations in order to coordinate sleep and activity patterns.

The work shows that nodes within the circadian network are sensitive to brief changes in temperature, and show that particular neurons are inhibited by heating and excited by cooling. It seems also that light and temperature are processed in distinct ways in the clock neutron network.

Interested to see the use of a fluorescent protein tool called CaMPARI that photo-converts from green to red in proportion to Ca2+ levels –  could this be used in plant work? Would require both light (photo-activation) and temperature manipulation…

The kinetics of temperature response was monitored by measuring intracellular Ca2+ concentrations using a calcium sensor called GCaMP6m and showed that particular neurons showed increases in intracellular calcium during cooling and decreases during heating.

The authors state that their findings reveal that the circadian network transduces brief and transient temperature changes and prolonged increases in temperature in distinct ways.

Thermoreceptors are found in structures in the antennae, called the aristae. Each arista contains both cold-sensitive and heat-sensitive cells. From their figure (below), they found that the responses to cooling and heating were attenuated when the aristae was removed.

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This work is interesting to use since we are trying to understand how plants respond to everyday changes in temperature – both short-term (daily fluctuations) and long term (seasonal) changes in temperature.

We are Stu-Dents

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Always amuses me the infographic from Matt Might called ‘The illustrated guide to a Ph.D,’ (click here for the full infographic page). Very useful for new Ph.D. candidates, but it I guess it’s just as valid for anyone working at the coal-face of scientific research. We are all just trying to make small dents at the boundary of scientific knowledge – ‘suppose that makes us all eternal stu-dents!

 

Friday read

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Science is a pretty earnest endeavour, sometimes it’s healthy to poke fun at it. The Independent Standard is a serious sounding web-site, but it’s effectively along the lines of the ‘Daily Mash‘ or ‘The Poke‘ for the Sciences.

I would recommend visiting this site if you want to cut-free from the Science wheel for a while. They are all spoof stories but sometimes the parody is so deep that it actually makes you think about the process of Science.

Some good examples are:

Human Tears Are The Best Thing For Precipitating DNA…..worth thinking about the next time a DNA/RNA prep comes around

An ‘Eppendorf For Life’ Scheme Launched…we have shopping bags for life…what about Eppendorfs for life..

and Leeds Scientist Refuses To Do Southern Blot….reporting regional variations in attitudes to molecular biology techniques

 

Collecting Cobras 2

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As a follow-up to the earlier ‘Counting Cobras‘ post, Hugh pointed out this recent opinion article from The EMBO Journal written by Alain Prochiantz.

The author recommends playing the science ‘game’ – for example by trying to publish in the so-called high ranking journals even if they do not necessarily reflect novelty and importance, because this is important for obtaining grant funding. Interestingly the idea of developing side projects appealed to me – the concept of planting seeds that, although would probably not get published in stellar journals or even accepted by peers short-term (see “You should stop science”, “you are making a fool of yourself”), but nonetheless with time might grow and branch out into research with high impact.

Other notable comments were the idea that “…there never really is a golden age [in science research]” and ” …it was not better yesterday but [..] it will be better tomorrow, provided that we never forget to defend the “Value of science”.

and finally…”Science remains a game, a game that must be taken very seriously, but nevertheless enjoyed”.

I suppose it’s trying to keep that balance – to play the game enjoyably!?

Coolit

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I’m liking the logo on my ice bucket today….what fun typefont is that?!

‘Polishing’ my RNA today – thats when we remove contaminating genomic DNA that might have co-purified during the RNA isolation stage. I’m using TURBO DNase from Invitrogen (…or is it ThermoFisher?)

In this case RNA:good :-), DNA:bad 😦

We don’t want DNA since it might interfere with measuring gene expression levels later on.

Clockwork (Orange)

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One thing that I find increasingly challenging is actually getting into the lab. A combination of major rebuilding a the University of Glasgow and Glasgow City Council imposing a ‘ring of no-free-parking steel’ around the West End of the city results in a unseemly work-day scramble to get the few remaining reasonably cost-effective parking spaces near the University.

Thus I often find myself zig-zagging across the city to eventually end up back across the Clyde river to do park-and-ride with the Glasgow Underground (aka the Clockwork Orange) at either the Shields Road or Kelvinbridge stations to get back over to the West End.

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I suppose I shouldn’t complain too much, however. At least I get to see this great mural of Glasgow’s West End by Alastair Gray, author of Lanark. The mural adorns the entrance/exit foyer of Hillhead underground station – the station nearest the University of Glasgow main campus.

The Bower Building – where the Glasgow collaborators are based – can be spotted on the mural map ! Here it is – located right next to the modern Woolfson Medical School Building:

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Collecting Cobras

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Interesting article last week – “performance-driven culture is ruining scientific research‘ – in the Guardian that has led to much discussion amongst the post-docs. All about the impact factor (IF) metrics – an arbitrary measure of how high (…or low) a journal is ranked. The article argues that a fixation on IFs, together with the Research Excellence Framework (REF) excise, detrimentally affects the diversity of Science research. The article starts off with an interesting anecdote about collecting cobra snakes during British rule in India…

Interesting also to see the range of comments after the article, ranging from – Science should be no different from other areas of work where performance led metrics are used  – to there needs to be a change in culture, would Darwin’s or Einstein’s ideas have emerged in a performance led scientific culture? Oh, and someone else noted the cheesy stock photo (above) as a common sight in all labs!

 

Let there be light

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Often we need to measure light intensity for our experiments and we use this little gizmo to do the job – a LI-COR light meter. Measuring light intensity is quite complex, but I find this explanation from GroWell (Hydroponics & Lighting) to be very useful:

Photons are counted in micromoles (µmol). One µmol is 602 quadrillion photons! I can’t even imagine a quadrillion!

I took a measurement of outdoor light intensity in Glasgow in February – reasonably bright, fast moving clouds, occasional cloud cover, temperature around 4oC. As you can see there were around 160 x 602 quadrillion photons hitting the sensor per second.

When there was no cloud I got readings up to around 600 umol – light intensity really does rapidly change….moving to the shade of a building I recorded around 45umol

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When we grow our plants in environmentally controlled growth cabinet we set the light intensity to around 130-150 mol. I suppose this is equivalent to a reasonably cloudy day in Glasgow! Plants will be used to much higher levels of light intensity though….how do they adapt to such rapid shifts in light?

 

 

RNA day

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….a day for making lots of RNA. Using Qiagen’s Plant RNA kit with their lilac and pink tops.

A tad whiffy due to using beta-mercaptoethanol – hope my lab colleagues don’t mind too much.

Follow the Qiagen protocol pretty much as is, except for the bit where you add ethanol to the QIAshredder flow through.

Instead of adding 96% ethanol, we routinely add 70% ethanol – see point 4 in the picture below. This seems to result in a better yield and quality of RNA. Our samples are for Arabidopsis plants (quite old….around 5 weeks old) and sometimes these plants have experienced constant light so they have lots of polyphenols.

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