SURF Presentations at RU

Although my SURF award was not a part of the SURF awards given out through Radford University, I was included in every event throughout the summer with the Office of Undergraduate Research. On October 10th, all of the SURF recipients were given an opportunity to present their research completed throughout the summer at the Summer Research Celebration at RU. During this event, students were given a choice as to whether they would like to orally present their research and findings, or if they would rather present a poster displaying their work. In order to continue preparing for my poster presentation at ASPB 2018, I chose to prepare a poster to present. Below are pictures of my poster, and Dr. PD and I during the presentation. It’s always a fun experience to be able to share my work with others, especially with many of my current and previous professors, along with numerous students. During the presentation, I even had a first-year biology student ask me to meet with her to discuss options that she has in our major.

Screen Shot 2017-10-18 at 9.41.52 AMposter pic

Wrapping Things Up

The last few weeks of my SURF were somewhat scattered out. Between my family going on vacation, and Dr. PD’s family doing the same the week after, it was hard to find a certain time to wrap things up. The last time I posted a weekly update, we were bulking up our AS1 cloned samples for sequencing. During our last week of research this summer, we successfully prepped the samples for sequencing, and sent them over to Virginia Tech, where the sequencing could be completed. Our results came back within a few days, however they were not exactly what we had hoped for. When cloning a plasmid, it often occurs that the clone is not in the correct orientation (the DNA sequence reads back to front, rather than front to back like normal). Of course, this is what happened to our AS1 clone. The good news is, there are ways to correct this! Given that this occurred on the last week of our SURF project, we plan to continue working throughout this semester in order to properly clone our plasmids in the correct orientation. Our plans are to find certain restriction enzymes that will “cut” right at the beginning of the plasmid sequence, and right at the end, allowing the plasmid to be inserted into the vector correctly. While we are continuing our research, and preparing for numerous presentations throughout the year, I will continue posting updates through this site, keeping you informed up until the ASPB 2018 in Montreal this summer!

Week Six: Moving Forward

Last week, in the midst of prepping the AS1 clone for sequencing, we had also started an AS2 ligation. Now that we fixed the little glitches from last week (now using cleaned PCR), we ended up with ten colonies on our AS2 plates.

Lewis, Tayler 2017-06-13 AS2 plates conc

Lewis, Tayler 2017-06-13 AS2 plates

After completing the process of inoculating the cultures from the colonies, isolating the plasmid DNA from the bacterial cultures, and digesting the plasmid, we ran a gel in order to check for the double bands (one band for the plasmid, the other for the DNA insert). Lewis, Tayler 2017-06-16 12hr 49min

The bands on the gel were very faint, and it was hard to determine if our DNA inserts were present. Because of this uncertainty, we decided to set up PCR reactions, using each corresponding plasmid as the template DNA in the reaction. If bands are present when we run the gel for the PCR products, that means that the DNA insert is present (there would not be a band if the DNA insert was not present due to the lack of DNA in the reaction). Lewis, Tayler 2017-06-21 13hr 26min

In lanes two, three, five, eight, and nine, there are bands present (lane one being the marker)! This is a good indicator that we do have DNA inserts in these samples. However, lane nine is very faint, so we decided to disregard that sample. Continuing on, we will use the four samples that gave us a clear band, and we will start to prep these for sequencing as well (sequencing determines the precise order of nucleotides within the DNA molecule, in this case a plasmid, in order to verify that the clone is in the correct orientation).

This week, we also set up PCR reactions similar to what we did last week; We set up three different reactions, the AS2 reverse and AS2 forward primers (positive control) , AS2 reverse and T7 reverse primers (negative control), and AS2 reverse and T7 forward primers, this time using AS2 as the template DNA instead of AS1. In total, we had twelve different PCR reactions, the 3 different set ups for each 4 plasmids. Below are pictures of the gel that we ran for these PCR reactions.

In lane one for each gel is the marker.
In the left image, lanes two-four are the PCR’s for one AS2 plasmid “1”, 6-8 for plasmid “2”, and lane 10 is a positive control. In the right image, 2-4 is for AS2 plasmid “4”, 6-8 for plasmid “7”, and lane 10 is a positive control.

While there are very present bands, the gels did not help us verify if the plasmids had the DNA insert, because the positive control, negative control, and the AS2 reverse/ T7 forward primer lane all had bands. This could be error caused by many different things. Next week, we will inoculate more cultures of the AS2 plasmids that showed bands on the first gel, in order to bulk the samples, ultimately to prep for sequencing.

Our wild type, AS1 mutant, and AS2 mutant plants are still continuing to grow (pictures taken just from this week)!

Left Column: Wild Type
Middle Column: AS1
Right Column: AS2

 

 

Week Five: Learning As We Go

Do you know that feeling you get when you realize something that you did, that you know you weren’t supposed to do, but didn’t catch it until it was too late? I had a few of those this week. They were simple mistakes, but they are also the reason that we haven’t been getting as much growth on our plates as we would like. I had previously mentioned that when you run a PCR, you must also clean it afterwards to remove all of the primers and other junk that you put in with the plasmid. When setting up a ligation in order to grow colonies on plates, you must use this cleaned PCR in your reaction. This brings us to our first mistake; I have not been using the cleaned PCR. This explains why we have not been having much growth on our AS1/AS2 plates. It is hard for colonies to grow with all of this other “junk” mixed in with the PCR.

Mistake number two: When setting up the “uncut” plate during a ligation, I have been using the plasmid that was digested with a restriction enzyme, meaning that the enzyme I used has cut the plasmid. You would think that the word “uncut” would tell me that I should not use the plasmid that has been “cut”. After resolving this issue, this is how our “uncut” plate looked.

lewis-tayler-2017-06-13-as2-plates.jpg

Other than fixing my mistakes this week, we also focused on determining the orientation of our AS1 clone! In order to do so, we took two different approaches. To begin, we drew out what our vector map (pET-23a), including how many base pairs the vector consisted of, and found restriction enzymes that “cut” the vector multiple times. In a normal digest, we use BamHI, which is a non-cutter. Because it does not cut the vector, we only see one band on the gel (the plasmid). For an enzyme that cuts once, we would see two bands, two cuts we would see three bands, etc. The two enzymes that we chose to digest were XhoI (1 cut) and PstI (3 cuts). Below is a picture of the vector map that I drew, including symbols such as B (BamHI), X (XhoI), and P (PstI) for where the enzymes cut on the vector. Along with each symbol, the base pair number in which they cut are also present. From B (198) to B (230) is where the AS1 insert would be. Each “cut” is color coordinated on the map to show how long (how many base pairs) the cut should be. Vector map pET23a drawing

When we run the gel in this case, we would expect to see 4 bands for the PstI digest (bands being at around 32 base pairs, 822 base pairs, 198 base pairs, and 2,546 base pairs), and 2 bands for the XhoI digest (bands being at around 4,310 base pairs an 428 base pairs).

The other approach that we took in order to determine the orientation was to set up a PCR reaction using the AS1 clone as our template DNA. In a normal PCR reaction, you have 1 microliter of your template DNA, 1 microliter of a reverse primer, 1 microliter of a forward primer, 12.5 microliters of your PCR master mix, and however much water is needed to bring the reaction up to volume. In this case, we set up three different reactions: one being our positive control by using the normal AS1 forward and reverse primers, one being, hopefully, our negative control by using AS1 reverse primer and a T7 Reverse primer (if there is a band on this reaction, it shows that our clone is in the wrong orientation), and the last using AS1 reverse primer and a T7 promoter (this being where we hope to see our band).

We ran both the PCR reactions and the digests on the same gel below:Lewis, Tayler 2017-06-14 AS1 digests and PCR XP

In lane one (the very bottom) is our marker, in lane two we have our PstI digest (3 cuts), in lane three is our XhoI digest (1 cut), in lane four is our positive control PCR, in lane five is our negative control, and in lane six is our AS1 reverse primer and T7 promoter.

For the PstI digest, there are three bands present (because the 32 base pair band is so small, we do not expect to see it on the gel). For the XhoI digest, there are two bands present, both appearing to be in the correct locations, showing us a positive sign for the clone to be in the correct orientation. As for the PCR products, there is a band for the positive control, and one for the AS2 reverse and T7F primer, which is a good thing! Because there is no band on the negative control, more than likely our clone is in the correct orientation! Next week, we will continue prepping our clone for sequencing, and we will prepare our AS2 plasmids to determine if the DNA insert is present.

Also this week, we transplanted our wildtype, AS1, and AS2 plants into soil!

Left: Wild Type                 Middle: AS1 mutant               Right: AS2 mutant

Week Four: A Step in the Right Direction

In order to talk about the big accomplishment that I had this week, I first need to mention all of the things that didn’t work out. Do you remember me mentioning the growth on the AS2 plates from last week? Unfortunately, they did not have the AS2 DNA insert. After inoculating the colonies of AS2 (suspending the bacterial colonies in LB, a liquid media used to culture bacteria, allowing the bacterial colonies to grow), we then isolated the AS2 plasmid from these cultures. This will allow us to digest each plasmid, and run them on a gel in order to see if the DNA insert is present. Unlike a normal gel, where one “band” shows that the digest worked correctly, we will be looking for two bands in this case, one for the digest, and another for the DNA insert.

Lewis, Tayler 2017-06-07 as2

The picture above is of the gel that we ran for the AS2 plasmid from the bacterial colony. As you can see, only one band is present in Lane 2 (lane 1 is the ladder, which is smushed together just due to the time that we allowed the gel to run), so the DNA insert is not present. While this specific ligation did not give us any culturues with the DNA insert, we decided to set up an AS1 ligation, this time using a different dephosphorylated vector.

(Top left, negative control. Top right, positive control. Bottom left, uncut plasmid. Bottom right,AS1 7:1) The left picture is the normal concentration, and the right is concentrated.

As you can see, we had multiple colonies of AS1, 10 in total! We went through the same process, inoculating the cultures from the colonies, isolating the plasmid from the cultures, digesting the plasmids, and running a gel (hoping to see two bands present)! This time, however, our wish came true!

lewis-tayler-2017-06-08-15hr-00min1.jpg

In lane 3, if you look really closely, you can the second band we were hoping for! The whole row of bands on the top near the open wells represents the digest. The faint band on well three, near the thinner black strip from the loading dye (towards the right of the picture), represents our AS1 DNA insert! This being the first time I have successfully cloned anything, both Dr. PD and I were pretty excited! After I had calmed down a little, we then discussed how the AS1 and AS2 inserts can clone in two different ways: forward and in reverse (forward being the ideal direction). Next week, we will be following the protocol in order to determine the orientation of our insert, and prepping the AS1 clone for sequencing!

Along with the bench work that we have been doing, we also wanted to grow the three different plants that we have been working with: the Wild Type plant, the AS1 mutant, and the AS2 mutant. Before we can transplant the seedlings into soil, we first cleaned the seeds, and plated them in order to allow them to grow in the incubator (speeding up their growth due to 24-hour exposure to light, and a temperature controlled environment).

(Wild Type on the left, AS1 mutant in the middle, AS2 mutant on the right).

Next week, we will also be transporting them into soil!

Week Three: It Only Takes One

ligase pun

When talking about the cloning process, I’ve heard Dr. PD say, “It only takes one colony that has the DNA insert to work!” This is starting to make more sense to me as we continue on with the cloning procedure.

Now that we have a new ligase to work with (as mentioned last week, the ligase we were previously using had expired), the first step coming into week three was to set up a ligation reaction. In order to test if the issue we were facing last week was due to the expired ligase, we set up the exact same reaction. This time, we had growth!

(Top left, negative control. Top right, uncut. Bottom left, positive control (no ligase). Bottom right, positive control).

It seems that the ligase issue has been resolved, so our next step was to try to insert our DNA of interest, in this case, AS2. We chose to try AS2 first because based on our gel images from before, we had consistent bands for AS2. Meaning that we definitely have a plasmid that has successfully been cut by our restriction enzyme. Here is a link to a video that will fully explain how an electrophoresis gel works. https://www.youtube.com/watch?v=XSO4ZBzu4jA

Using the new ligase, here are our reactions that we set up:

(uL= microliters)

Neg. Control: 

2 uL dephosphorylated vector

0.5 uL ligase

1 uL buffer

6.5 uL water

=10 uL

Pos. Control: 

1 uL phosphorylated vector

1 uL buffer

0.5 uL ligase

7.5 uL water

=10 uL

3:1 AS2: 

1 uL dephosphorylated vector

3 uL AS2 PCR

0.5 uL ligase

1 uL buffer

4.5 uL water

=10 uL

7:1 AS2: 

1 uL dephosphorylated vector

6.5 uL AS2 PCR

0.5 uL ligase

1 uL buffer

1 uL water

=10 uL

After the ligations had been plated, I jokingly had a little talk with the plates in the incubator. “Please, please, please. GROW!” This little pep talk must have had an effect, because the next morning, we had colonies!

(Top left, 3:1 AS2. Top right, negative control. Bottom left, positive control. Bottom right, 7:1 AS2).

These images of the plates were taken under UV light, each having a label describing what the plates contained. The image on the left contained the “normal” concentration reactions, as to where the image on the right contained the “concentrated” reactions. The only difference between the two is the amount of liquid that the DNA was suspended in (concentrated having less). The little white dots on the plates indicate individual colonies. The 3:1 and the 7:1 ratios contained the same substances in the reaction setup, however the 7:1 ratio received more of the AS2 insert.

As you can see, there were few to no colonies on the negative control. The few colonies on the negative control are not necessarily a bad thing, considering the number of colonies on the positive control were wayyy higher. This is not a bad ratio between the two controls. If there were much more colonies on the negative control, then we would be worried. On the 3:1 plates, both concentrated and normal, we did not have much luck. However, on the 7:1 plates, both the normal and concentrated plates had 2-3 colonies. Not as much as we would prefer, but this is a starting point!

In between ligation reactions, we have still been doing DNA prep, such as bulking up our PCR products, making solutions, pouring plates, etc. Also this week, many of the other SURF students through RU and their mentors, along with myself and Dr. PD, all got together for donuts and coffee one morning. It was nice to just sit around, chat a little bit about our research, and just hangout. Next week, we will be checking to see if our colonies on the AS2 ligations plates contain the insert DNA!

 

Week Two: Our first setback

cloning pun.jpg

Going in to week two, I was super excited to start the cloning process! By cloning, I mean cloning our AS1 and AS2 genes in order to create a large number of copies of each gene. The cloned DNA can be used for many different things, such as helping us look at how mutations may affect a gene’s function, and to figure out the specific function of the gene of interest. These things can ultimately help us to verify our 3D computational models (HOPEFULLY!!). The first step in the cloning process is DNA preparation, which is what I talked about in last week’s post (running gels, PCR, making solutions, etc.). After the DNA is prepped and ready to go, the next step in the cloning process is to complete a ligation. A ligation is when you join two DNA strands together by using an enzyme called ligase. The ligation will form together our vector (a DNA molecule used to carry foreign genetic material into another cell) and our gene of interest (AS1 or AS2). In our first attempt, we set up 5 reactions, one being a negative control, 2 of them being different concentrations of AS1, and the last 2 being different concentrations of AS2. After setting up the reactions, we placed the ligation mix on LB + ampicillin plates, incubating the plates overnight at ~40 degrees Celsius. The next day, there were no colonies on the plates. The next step was to actually take a step back, not to focus so much on adding the gene of interest (AS1/AS2), but to set up a reaction of only positive and negative controls in order to see, basically, what’s working and what isn’t. In our second reaction setup, we used a different ligase brand than the first reaction, in order to see if that was the issue. We set up four reactions: Uncut, negative control, positive control, and positive control (no ligase). The reactions are listed below.

(uL= microliters)

Uncut

1 uL diluted plasmid (pET23a B 5: 1/10 dilution)

1 uL 10x buffer

8 uL water

=10 uL

-Control

1 uL dephosphorylated. Vector

0.5 uL ligase

1 uL buffer

7.5 uL water

=10 uL

+Control

0.5 uL phosphorylated vector

1 uL buffer

0.5 uL ligase

8 uL water

=10 uL

+ Control (no ligase)

0.5 ul phosphorylated vector

1 uL buffer

8.5 uL water

=10 uL

The “uncut” reaction was only given a plasmid (an independent, self-replicating DNA molecule that carries only a few genes), meaning that there should be no trouble with having growth on that plate. The negative control was not given anything for the dephosphorylated vector (removal of phosphate groups from a DNA fragment in order to prevent ligation) to bind to, so we expect no growth on that plate. The positive control was given phosphorylated vector (still carrying the phosphate groups) and ligase, in order to see if our vector can “stick” back together. The positive control without ligase was given a phosphorylated vector with nothing to relegate to, meaning there should not be growth on this plate. After letting the plates sit ~ (about) 40 degrees Celsius overnight, the next morning we checked for growth. There was nothing. Looking at the bright side, its good that we didn’t have growth on the negative control or the positive control without ligase, however the other two plates, not such a good thing.

So, the main thing I learned this week was that most of the time spent doing lab and bench work, is actually just troubleshooting what could have gone wrong. Here are examples of LB + ampicillin plates. The plate on the left has no growth, and the plate on the right does. “Growth” on a plate represents individual colonies of bacteria. When we add the gene of interest (AS1/AS2) into the ligation, each colony (or most) should contain that DNA segment.

 

So basically, attempts 1 and 2 looked exactly like the plate above on the left. Well actually, the plate on the left is a picture of my actual plate used during the “unsuccessful” ligation. There was no growth. This could be caused by a number of things: the ligase we used has went bad, human error is a possibility, or even the concentration of the vector was not the correct amount. Given that Dr. PD said that this was completely normal, and that her suspicion is that the ligase has expired, we borrowed another professor’s ligase and buffer (the two have to be the same brand and made by the same company), and we will see next week if there is growth on the plates!