DUN DUN DUN: DIATOM TRANSFORMATION (video edition)

I TRANSFORMED MY DIATOMS. ALL IS WELL. THIS IS AWESOME.

Well of course that is if we successfully transformed the diatoms. There's a 1-2 week grow period during which we have to wait in order to see transformed clones. I'll keep this blog posted and will post a text post regarding the transformation soon as well.

Tumblr & Science

I've created a Tumblr page to post cool pictures from lab to run along side my blog here about my project and you should really check it out:

This is where I post stuff like this that I've made:

But really, I should be posting cool stuff like that here as well, which I will do in the future.

Building inducible expression plasmids (aka Science)

Below is a video I've been trying to publish for a long time coming this summer, but because my summer project only came to a close this week, I've been hesitant to post anything.



In this video, I give an update on my lab work and discuss my overall lab project, which is a great review for myself and my viewers. This is an exciting time for me at Clark University, because I’m beginning the real stages of becoming a graduate student in our accelerated Master’s program. After conducting undergraduate research, I’m using it to support a 5th year of schooling at Clark to finish up my Master’s.

My project concerns the 3’ UTR of the nitrogen-assimilating genes nitrate reductase and nitrite reductase in diatoms (if you haven't read up on my blog before), because we have evidence that along this region these genes are up- and down-regulated by environmental conditions. To test this hypothesis, I’ve created plasmids that drive expression of a reporter gene (GFP) with promoter and terminator regions derived from diatom genes. We’ll compare the activity of GFP with differing 3’ UTR elements to determine whether this region of the nitrogen-assimilating genes is crucial in their regulation.

For the gene nitrate reductase, this is old news. But it is news for my blog that I've completed the plasmids for nitrite reductase, which is really the story of my video.

I'm really excited to be where I am in my project. This week, I hope to genetically transform my diatoms with the plasmids I've created. Oh baby Darwin, this could be a HUGE week for me and my scientific career! I'll be back soon with transformation news.

The completion of my NiR plasmids

This summer's project has been to complete inducible expression plasmids to test the regulation of nitrite reductase (NiR) in the diatom Thalassiosira pseudonana. Inducible expression means we can induce the expression of a gene located on a plasmid based on environmental factors. These plasmids will be used to test whether the 3' end of the NiR plays a role in regulation the gene's activity based on environmental cues. We'll compare the activity of our reporter gene (GFP) from our control plasmid (with an NiR terminating region) and our developing experimental plasmid (with an Actin terminating region, as actin has no role with nitrogen assimilation).

An analytical double digest and gel to determine which of my bacteria colonies actually had the plasmid I wanted. My colony screen gave me some funny results, so I wanted to double check with a digest. The digest confirmed several of my colonies appear to have the plasmid and correct insert, giving me the green light to grow up more bacteria cultures and prep and purify their plasmids.

While it appears my NiR-NiR plasmid is completed (sections of the NiR gene flank the reporter gene in the plasmid), the completion of the experimental plasmid is right behind the control plasmid. Today I transformed some bacteria with what I hope is the complete experimental plasmid. I should know by tomorrow afternoon if I have what I think I do!

Meanwhile, I have scheduled a date at the University of Rhode Island to transform my diatoms! While I may only transform the diatoms with my set of nitrate reductase (NR, a different but related gene) plasmids (both with an NR terminus and an actin terminus, like the NiR plasmids I'm finishing up now).

This DNA has a sequence and I have every intention to find out what it is! (and other stories)

In my quest to amplify a region of the nitrite reductase (NiR) gene that I can then ligate into a plasmid for in vivo testing, I needed to sequence my portion of DNA for three main reasons:

1. I needed to make sure that the restriction sites I added with gene specific primers were added to either end of the sequence. These restriction sites will allow me to cut out my amplified piece of DNA that is cloned into a cloning vector.
2. I need to verify that the sequence of DNA I've been cloning is indeed from the NiR gene, especially since the DNA sequence has been marginally longer on my agarose gels than anticipated.
3. Previous data acquired from a database told me I should be able to get a restriction enzyme to cut somewhere in the middle of the DNA sequence I had cloned, but the restriction enzyme wouldn't cut in the middle (but it did cut on either side of the DNA sequence that I cloned into a vector, as the vector had that restriction site on either side of the multiple cloning site). Essentially, that restriction site didn't exist in my DNA sequence like it should have.

So, couple weeks ago, I performed a PCR cycle sequencing reaction to start the process of sequencing. The purpose of this sequencing reaction is to amplify a region of DNA with DNA bases that have special tags. These tags are read by a sequencing machine yielding a series of base signals like below:

The color coded lines refer to a single base category (either A, T, G or C) and denote the signal strength at that spot in the sequence. Based on the predominant signal at a spot in the sequence, the software makes a judgment call as to which base is present at that location. Often it is quite clear to the machine and software that a particular letter of DNA is certainly an A at a specific location because there is a single strong signal peak. Other times there may be several signal peaks at a single location because of a lower quality sequencing reaction (e.g. there is too much or too little DNA to be read properly), which make it harder to determine which DNA letter actually exists in the DNA sequence.

In order for the DNA to be read by the sequencing machine, it needs to be single stranded. This means we make up separate PCR reactions with only one primer, which means that the one primer in a PCR reaction will attach to the DNA and copy the DNA until it falls off. To make it easier on myself, I followed a standard protocol that sequences a section of DNA that has been cloned into a plasmid vector. This way there is a buffer of vector plasmid sequence on either side of my DNA sequence of interest, where the primers that pair with the vector plasmid would bind.

I performed my PCR reaction and prepared it for the sequencing machine and was about to load my DNA onto the DNA sequencing plate (which goes into the machine), when I was notified that the machine was down and I couldn't sequence that day or possibly for the rest of the week. It took me a week or more for my samples to get read, but they finally did get read and my sequences were good enough to give me some answers.

I use a special computer program that will align the DNA sequences to one another. Running multiple samples of the same sequence will allow the program to compare answers and yield a single, best result sequence. Interestingly enough, while my four samples all matched each other, there was a small region (about 20 bases) that did not match up with the database sequence for my gene. This may have been why my restriction site wouldn't cut--because it wasn't there. One of my labmates is going to sequence the same gene soon, so I will be able to compare my results with hers and determine whether the predicted sequence in the database needs to be updated.

I can then take the sequence from the first program (the results) and pop it into another program, to quickly find all of the restriction sites that are present.

Here I can see that my BamHI and NotI restriction sites are indeed at either end of my sequence, with the EcoRI sites just outside of the Bam and Not sites. Because the EcoRI sites are in the vector which I cloned my sequence with, I know that the Bam and Not sites are on either side of my sequence like they should be.

At present I have ligated this section of DNA (the piece of the NiR gene) with the other portion of the NiR plasmid, and will transform some bacteria today. If the plasmid worked and was successfully cloned into my cells, they bacteria will be able to grow on the plate laced with ampicillin. Here's hoping!

Clarkies on college life

Ever wonder what life at Clark is like? The five video series I created attempts to answer the beginning of those sort of questions.

Why should students pick your major?
What was your favorite class at Clark?
What is your favorite restaurant close to campus?
What is your favorite campus event?
What is the one thing you look forward to all year?

General campus lolz

My girlfriend and I had dinner on the green the other night, and it was really pretty. But then this squirrel started getting a little close and started to creep us out.

My little friend got a little close for comfort.

This is after I posted this video to my YouTube channel:

On an unrelated note, I made this Rage Comic last week, which I think sums up my summer pretty well:

Also, more dry ice:

How to reuse plastic pipet tip loaders

Working in a biology lab, you may have discovered the wonders of boxed pipet tip packs that allow you to quickly load multiple boxes of pipet tips. These boxes of pipet tips are made up of multiple layers of tips stacked on top of each other, separated by plastic sheets (below), which are left behind once you finish a box of pipet tips. I've been collecting these sheets for quite a while and have since found a use for them.

Plastic tip loader sheets, henceforth called plastic sheets. They need a better name.

With some ordinary lab tape, these plastic sheets can be made into handy baskets used to separate lab supplies. By stacking and taping the sheets in different orientations, you can make custom shaped baskets that accommodate tubes, caps, or anything else your lab has generous quantities of.

At first, I made dividers--a square of sheets taped together--that allowed the separation of caps from tubes in our messy lab tube drawer. While this made the messy lab tube drawer a little tidier, I found that making the dividers into baskets (by giving them a bottom) greatly increased their efficiency because I could pick up and shuffle the order of the tubes and caps within the drawer.

These plastic sheets can be made into a wide variety of different shapes and sizes of baskets. Overlapping the plastic sheets or carefully cutting off a section of a sheet allows you to customize a basket to fit those awkwardly shaped tubes or hold your collection of caps that has gotten out of control.

While you could probably figure it out yourself now that I've given you this awesome idea, and there are definitely a ton of ways to make these baskets, below is a general step-by-step guide to make these crafty creations.

Supplies needed in order to create these crafty creations:
1.) Six or more plastic pipet tip loader sheets.
2.) Tape (I used lab tape because it is handy, fairly strong, and comes in a variety of colours).
3.) A cutting device (most people use scissors... which I recommend. Razor blades cut thumbs, but of course I wouldn't know that first hand).

Directions:
1.) Decide the size of the basket you need. I'm making a basic 1 by 1 sheet basket to hold tube caps.

One sheet by one sheet (these will be the side of the basket).

2.) Because these sheets are rectangles, the bottom of the basket will most likely have to be cut and taped together to make a square. I do one of two things:

Top: you can overlap two sheets to match the length of the basket side.
Bottom: you can trim the second sheet to match the length of the basket side, so the bottom of the basket will lay flush instead of having a slight bump.

I'm going to go the second route and use the spare green piece of plastic sheet to make my bottom. The top option (overlapping sheets) is stronger, but won't sit evenly on a flat surface.

With the bottom of the basket sized up, it's time to start taping this bad boy together. Professor! Where do we keep our tape?

3.) Tape the basket bottom together. Be sure to put tape on either side of the sheets.

4.) Tape the four sides of the basket onto the bottom. I do NOT tape the outside of the bottom to each side just yet, because the tape usually rips. I tape the bottom to the sides once the basket is erected.

5.) Tape the sides together. First, pull the sides up, perpendicular to the bottom.

Go top left to top right, to bottom left to bottom right.

Then place a piece of tape on the inside of the sides where they meet. Cut the tape from the top down to the edge of the basket and fold the pieces of tape over. Repeat this step by placing a piece of tape on the outside of the basket sides, cut the tape, and fold the pieces of the tape toward the inside of the basket. Both the inside and the outside of the basket corner top should be covered in tape.

Two sides taped to one another.

6.) Tape all four of the sides together. Now your basket is (essentially) completed.

Looking pretty spiffy!

7.) To increase the integrity of the bottom of the basket, a piece of tape along each edge of the bottom taping the bottom to the side of the basket is a good idea. The easiest place to tape is on the outside; if you really want the bottom of the basket to stay attached the sides you can try taping the inside of the bottom to the sides as well. You can also put more take along the corners where the sides of the basket meet each other.

The yellow, red, pink (which isn't very pink), and orange pieces of tape keep the bottom of the basket attached to each side of the basket.

For a more professional look:

- Try overlapping the plastic sheets to make a customized fit rather than cutting sheets and taping them together. Less tape may look better.

- Cut the edge of your pieces of tape for a flat edge instead of the standard wavy edge you get from the tape dispenser.

- Use only one color of plastic sheet and stick to only one color of tape (I hope you already thought of this.)

> > Feel free to e-mail me with questions and comments.

A quiet day in the lab

Sup July? SUP SUCCESSFUL TRANSFORMATIONS?

I've been having trouble transforming E. coli cells with my plasmid vector, within which is a small DNA fragment (my NiR terminator) that I will want to restriction cut out. By transforming bacteria with the plasmid vector, I'll make additional copies of the plasmid and be able to freeze and save the plasmid for later use if necessary.

My lab mates and I spent several weeks trying to figure out why our transformations were doing so poorly and why we were receiving such low plasmid yields from transformed bacteria. I myself figured out that one problem was the ampicillin used to make the agar plates upon which we grow our bacteria had degraded over time, and that the antibiotic was not selecting strongly enough to weed out bacteria with plasmids and bacteria without plasmids. This is the reason why we were not getting good plasmid yields and another reason why our bacteria were not growing when transferred from "old" plates to new agar plates with freshly made ampicillin.

We also concluded that the bacteria cells we were transforming were not up to par to yield the results we needed, so we ordered some new transformation kits.

But in order to successfully clone PCR product into a plasmid vector to transform into bacteria, the PCR product needs to be freshly made. In order to get new PCR product, I re-amplified older PCR product in the same reaction I ran before. I ran four different reactions using the PCR DNA in four different DNA concentrations: 1:1, 1:10, 1:100, & 1:1,000 (lanes 2, 3, 4 & 5 in the picture below respectively). This way I can determine which reaction had too much starting DNA and too little. After my reaction, I ran part of it on a gel to see how each reaction went. I definitely got much larger yields in the 1:1 & 1:10 dilutions (there was probably too much DNA even), so I used the second dilution (1:100, lane 4) to clone into the plasmid vector.

I used PCR product from lane 4 to clone into a vector plasmid for the transformation.

Using the vector plasmid, I transformed them into the bacteria and let them grow over night on an agar plate. I then performed a colony screen, which is a PCR reaction using single bacteria colonies to supply the DNA. That PCR reaction yielded the below gel:

While this is a slightly messy colony screen gel, several of these colonies should suffice!

What we're seeing in this gel is the molecular ladder at the top and then 10 different colony screen reactions. They're pretty streaky, which is probably because there was a lot of bacterial DNA in each PCR reaction. What I wanted was a single band at around 700 basepairs, which is roughly half way between the two second most right bands on the ladder. As such, lanes 4, 6, 7 & 8 are good candidates for colonies that have my plasmid with the correct insert.

BRB time for the holiday weekend!