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).

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!

Sometimes science isn't linear (and I'm not talking about exponential growth)

Sometimes science (my project) isn't linear, and I've been getting caught up in this recently with my posting.

I wanted to do a series of posts and videos on the process of transformation and the completion of my first NiR plasmid, but what I found was that sometimes things don't work out the way you want.

So, let me backtrack a bit.

As I mentioned before, it appears I have transformed bacteria colonies that have my PCR insert. Great! But I've been having trouble getting the insert to amplify out of the plasmid once again. I grew up several bacteria colonies that looked like they had my insert (white colonies on X-gal) and performed a plasmid prep that yielded very little plasmid DNA. I need a decent amount of this plasmid to allow me to digest (cut) out and obtain the insert.

Now, I'm trying to do yet another PCR reaction in a much larger volume (50µl rather than 10-20µl) using the plasmids I obtained from my lame plasmid prep. If this works, I'll have a lot of copies of the NiR terminator insert, which I can then slice off the ends with restriction enzymes. Then, the insert would be ready for the next step.

That is, if I can get this to work. :-\

Sometimes Digests Possess The Answer

Today I'm running a digest on a plasmid in an attempt to cut out a portion of DNA.

This plasmid is the nitrite reductase (NiR) gene terminator region cloned into a vector (called pCR4). Just outside of where the terminator region should be inserted is a series of primer points and restriction sites.  EcoRI restriction sites flank just outside the cloning site, and as such I'm running an EcoRI restriction digest. The digest only takes an hour at 37°C on a heating block (right), and is a simple reaction containing just restriction enzymes, buffer, DNA, and water.

I took a quarter of the restriction digest reaction (5 of the 20µl), added 1µl loading dye, and ran the reaction on a 2% agarose gel:


Here we can see in the second lane my digest. The really bright band is the vector (most of the DNA in the plasmid) and the smaller band (further along on the gel) in my insert, just under 1,000 base pairs. The ladder (the top and 5th lanes) is a 1kb ladder--moving right to left, the smallest band is 500bp, then 1kb, 1.5kb, 2kb, 3kb, and 4kb, etc. I like to use a 1kb ladder most of the time because it's really easy to use, and I can quickly tell which band is which because the 3kb mark is the brightest in the ladder. in my gel here, the vector sequence is larger than 3kb, and roughly equal to the 4kb band (which was expected). The insert that was digested out in the reaction is just short of 1kb (which was also expected.

I then ran the rest of the reaction on a second gel (which was a sort of a waste, but I always want to check my reaction before I look to do anything else with it), because my digest worked. I wanted to cut out the smaller band and use that in a PCR reaction, to make it easier for the primers to amplify them. Since I've been having trouble getting the primers to work on this plasmid, we decided we might as well try this. So I ran the rest of the digest reaction in lanes 2 and 3--you can see where I cut out the bands, which I did with a razor blade (which is the black silhouette on the right).

I then melted the agarose gel that I cut out with my DNA band, and cleaned up the solution. By binding the DNA to a small filter, I could clean the DNA and remove the gel. I then yielded (what I hope is) the digested plasmid insert. I'll use this cleaned up insert from the gel in a PCR reaction overnight tonight and hopefully get some better results.


Below is a cartoon representing the NiR sequence I'm working with. For now, I'm just focusing on the terminator region, which is the 3' untranslated region (UTR) sequence just after the stop codon. By sequencing the DNA we're working with or looking up the desired DNA sequence online at a databank website, we can model the DNA sequence and figure out where the start and stop codons most likely exist. We can then make primers to amplify specific regions along that DNA.

The blue rectangle represents the NiR terminator that we're amplifying: it's just a little bit longer on either side of the actual 3' UTR, which means we're sure to amplify the entire 3' UTR.

More Plasmid Work

I've already talked about how I've made an inducible expression plasmid to test the mRNA stability of nitrate reductase (NR) transcripts in vivo in the diatom Thalassiosira pseudonana. The plasmids run by using a promoter and terminator sequence to run the expression of a reporter gene (we're using GFP). We have a set of NR plasmids, one with a terminal region of NR and one with a terminal region of action (which has nothing to do with nitrogen assimilation). The former plasmid should mimic endogenous activity, whereas the latter plasmid should not mimic the normal conditions in the cell. NR reduces nitrate (NO3-) to nitrite (NO2-), which is then reduced to ammonium (NH4+) by nitrite reductase (NiR). As such, in the scheme of nitrogen assimilation in diatoms, it makes sense to test the regulation of NiR as well.

To do this, I've started work on a set of NiR plasmids. They will also run the inducible expression of GFP, but with their own promoter and terminator regions. Both will have their NiR promoter region, whereas one will have the NiR terminator and one will have the actin terminator region. Both sets of plasmids will be transformed through particle bombardment and then in vivo expression can be measured (through GFP activity).

As it stands, we have a plasmid that contains the NiR promoter & GFP but not the proper terminator. The NiR terminator was cloned into a separate plasmid as part of the process in amplifying out the terminator region of interest. Currently, I'm trying to amplify the terminator out of the plasmid, and in doing so add restriction sites to the plasmid.

The cartoon below represents the series of steps that we have to do in order to manufacture the desired fragment of DNA with restriction sites at the beginning and end of it, which allow us to easily cut out the DNA fragment and place it into a plasmid. Right now, the NiR terminator is represented by the red box. It was amplified out of the entire NiR gene to yield a small piece (between 500bp and 1kb) of the the gene. This piece was at the very end of the open reading frame and extended past the 3' UTR (the terminal region). This was done in a PCR reaction, and the PCR product was inserted into a vector, transformed into bacteria, grown, and then the plasmids were isolated once again the yield the below plasmid.

What I want to do is amplify a smaller portion of the insert out of the plasmid, and make a bunch of copies of it through a PCR reaction. A second set of primers (the green lines) will amplify within the region of the insert, while adding restriction sites. Currently, I've having problems getting this PCR to work because less than half of the primer fits to the DNA of the current insert, while the other half is going to add the restriction site. However, I changed the protocol for the PCR reaction I'm running at the moment. I changed the annealing temperature for the first 10 rounds of my PCR and then I'll bring it back up to what I ran it yesterday (a PCR reaction that did not work, lanes 3-5; right--below right my PCR samples loaded; the faint blue samples are the ladders I used, lanes 1, 2 & 8; the red samples are my PCR reactions that used Coral Load, a special PCR buffer/loading dye combination, lanes 3-7).

...NiR terminator amplification to be continued...