“More than 10% of currently described shark species are luminous.”
Apparently, while the hormone prolactin activates light in lantern sharks, it has the opposite effect in pygmy sharks, turning bio luminescence “off.”
Source: BBC Nature, http://www.bbc.co.uk/nature/17812363
New Zealand's glow worms have a glowing tail to lure prey. Image credit: Sir Francis Canker
Gallery – Glowing report: the mysteries of bioluminescence – Image 1 – New Scientist.
Some luminescent critters from New Scientist – fireflies, glow worms, mushrooms, and jellies! And of course, good ‘ol GFP in cats to test insertion of the gene for feline HIV resistance.
What does it mean to align a DNA sequence?
We send our samples to an external lab who takes our specially prepared samples and generates data files with the individual nucleotides that make up our gene. These are like letters in a book.
Our original gene has been published and is public. You can check out all the public genomes that have been sequenced at the Gen Bank.
I’m working on a gene from T. elongatus and have been changing individual nucleotides in its genetic sequence in order to generate mutations. Using this method I’m essentially taking a characteristic of T. elongatus and changing it slightly to get it to do something different in a controlled fashion.
In order to ensure that what I’m working with is actually the gene I want to be working with I send the DNA off for sequencing. When the data comes back I match it up with the published DNA data to make sure they match.
What you see if they don’t match:
The output shows 2 rows. Your sequence and the one you’re comparing it to. Depending on the application you use to do the comparison different symbols appear to show where the matches. The matches for the app I use shows lines connecting the matching locations. When you have a good match you’ll see something like this (I highlight my matches in yellow):
Over the last month our DNA sequencing has hit some strange snags.
It’s difficult to troubleshoot a process when you’ve been doing it the same way for so long and previously has just worked fine!
This time I cooked up some NZy broth to grow cultures in before mini-prepping them. The main difference between the broth we normally use and the NZY seems to be using NA amine and adding MgCl2 and MgSO4.
I also found a nice mac app to use to align the sequences in order to verify the DNA was definitely my gene. Check out Serial Cloner. It has lots of useful features.
Tomorrow I should be able to mutate! Wish me luck!
NZY+ Broth (per Liter)
10 g of NZ amine
5 g of yeast extract
5 g of NaCl
Add deionized H2O to a final volume of 1 liter. Adjust to pH 7.5 using NaOH.
Autoclave.
Add the following filer-sterilized supplements prior to use:
12.5 ml of 1 M MgCl2
12.5 ml of 1 M MgSO4
20 ml of 20% (w/v) glucose (or 10 ml of 2 M glucose)
Glowing bioluminescent plankton in the tide line washes up onto a beach on Vaadhoo Island, Raa Atoll, Maldives, with stars above and a ship’s lights on the horizon
Picture: Doug Perrine / Barcroft Media
So! Next round of mutation is coming up!
We looked at some protocols and decided that while we often use substitutions for various methods and materials this time we’re going to follow the instructions as closely as possible.
We’ll be using Agilent’s site-directed mutagenesis kit and going through all the control processes specified.
Normally we don’t do controls but since there have been some problems recently (growth, sequencing, possible mutations) I’m going to run through the procedures with the controls and do the double checking. The kit uses a pWhitescript control plasmid which we’ll use to transform into XL10-Gold competent E. coli cells. The XL10-Gold cells, if transformed correctly, should grow on a plate inoculated with ampicillin and look like small white-ish colonies. As an extra verification that transformation occurred as expected we can perform a test to see if the phenotype (blue in this case) is expressed on a media plate containing IPTG and X-gal. This might seem to be going overboard a little, but I think these are good methods to implement for this next round.
Before performing the mutagenesis I’ve streaked some LB plates with stocks from way back (verified non-contaminated with sequencing) to get template colonies ready. I made plates of 911T-C stored in DH5α and 911T-C LMG-194. Once I have colonies I’ll make 2 mL cultures in rich media broth and then mini-prep them to get naked DNA. We have a Nano-Drop 2000 in the lab which is great for determining concentrations. The nano-drop will give me the DNA concentration which I can use to calculate the how much material I should use for the mutation / PCR process.
I made NZY broth which we don’t normally use. It was a little tricky getting the pH correctly as the “magic wand” of our pH meter can be kind of wiley.
I hope my plates grow up nicely tonight!
Now to double check that all the pieces of the kit are present and then tomorrow – I’m going for it!
I was looking at graduate programs when I came across UCSD’s Biomedical Sciences page…and immediately recognized the beautiful rainbow of fluorescent proteins! Life is amazing!
Fluorescent proteins from UCSD's Biomedical Sciences Graduate Program site!
