OUTPACE 2018 FINALE

by Adetokunbo Ayokanmbi

All good things must come to an end. OUTPACE 2018 has been a fantastic research and learning experience. It also allowed us to meet new people who we may not have had the opportunity to meet without this great program. OUTPACE introduced many of us to new plant and biology concepts in a research setting. We were able to have hands-on experience working with various plants and pathogens. Overall, everyone was grateful for this opportunity to learn and broaden our scientific knowledge under Dr. Karolina Mukhtar.

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Our final day consisted of a going away ceremony for all the participants and TAs of the program. It was a fun celebration where we were awarded certificates for completing the program. After receiving the certificates, we celebrated with a special OUTPACE limited edition cake.

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At the end, Dr. Mukhtar gave us inspiring words about pursuing a science career, and she let us know that we are always welcomed to contact her if we need anything and all of us are a special group of students to her. OUTPACE 2018 has been an amazing experience for everyone involved, and we congratulate everyone for successfully completing this summer program and wish them well in their future endeavors.

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Outpace of Summer 2018 was another major success!

by Nick McRae

The Grand Finally was a head to head competition between this year’s students.

Name of the game was: VEGEVADERS – Plants vs Pathogens.

Game 1

Sponsored by the National Science Foundation, Vegevaders is an excellent game depicting the same mechanism of Pathogens Attacking and Plants Defending that we’ve come to understand though Outpace. The creaters of this game did an outstanding job at tying in the principles of bacterial infiltration and the plant’s detection & immune responses. It was a fun, & accurate way to understand the rather complex molecular processes of the constant battle between these two.

Board

Whoppers

As you can imagine – The Stakes Were High & The Pressure Was On!!!

Getting Serious

The winner would not only have bragging rights but walk away with the choice of premium candies…Ohhh yeah & the Whoppers were in very high demand! Haha

The Bracket was determined from best out of three, pitting students together; for a test like no other. Everything we’ve ever learned in college was all riding on this one moment…Lol

MVVP

Niharika walked away Victorious! The Official Most Valuable Vegevader/Player of 2018.

All in all, it was a lot of fun for everyone & a great way to wrap up our Outpace experience!

 

Fungal Infection 2

by Madison Lathem

For this lab we observed the plates of Arabidopsis leaves that were infected with fungal spores on May 21, 2018. Two varieties of Arabidopsis were originally infected with Botrytis Cinerea; Columbia or wild type and PAD2 mutants. PAD2 mutants are a species of Arabidopsis with several mutation that make it a knockout for several genes including genes that encode for immune response. This means that it was expected to be more greatly affected by the fungal spores and therefore appear sicker after incubation.

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Today we compared the two varieties of Arabidopsis based on how infected they appeared to be. The infection was rated by counting the leaves that were completely stressed, purple all the way through, as one complete infection and the leaves in which the stress was contained to the vascular system as ½ infection. The results were then graphed, and the varieties were compared. Surprisingly the Colombian variety appeared to be more infected than the PAD2 variety. This could be because the infection was not from the fungus we originally thought we infected it with, but rather a bacterium in which the PAD2 variety was resistant to.

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Garden Tour 2

by Madison Lathem

For this lab we went out to the UAB gardens to collect plant tissue samples that we believed had either a fungal infection or a bacterial infection. Each of us collected our samples and placed them in a Ziploc baggie, which was labeled with our name and the type of plant in which it was obtained from and carried them back to the lab.1

The plant tissue samples that were believed to have fungal infections were cut into tiny squares to be placed on a pre-made V8 media plate. Each student cut out two squares of tissue sample washed them in Clorox and placed them on the plates. The plates were then allowed to incubate for a couple days till fungal growth could be observed.

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The plant tissue samples believed to be infected with bacteria were taken to the cold room and allowed to sit and were processed the following day. The bacterial plant tissue samples were processed by cutting the tissue into tiny squares, washed with Clorox and adding them to a tube with 500 μL of distilled water and a metal pellet. The Tube was then taken to doctor Mukhtars lab and placed in the homogenizer until the solution was a homogenous mixture. The mixture was then diluted in three serial dilutions; 1:10, 1:100 and 1:1000. Each dilution was then spotted in onto three different media, YPD, V8, and KB, plates. They were then allowed to incubate to observe bacterial growth.

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Fungal Infection

by Nick McRae and Meghan Letson

Botrytis Cinerea is the necrotrophic fungal pathogen most commonly associated with Grey Mold on fruit. (often seen on strawberries going bad) It also happens to be a very common plant pathogen which attacks almost every plant species.

For this lab, we used to B. cinerea to study immune response of this fungal infection on Arabidopsis. To do this, we had to colonize the fungus on a V8 based Agar medium. We then collected and count spores from B. cinereal:

Spore Collection

After collection, we diluted to an ideal concentration. Utilizing gridlines we then could infer we were within the acceptable range to proceed to the next step:

Estimating Concentration

We then chose five leaf samples from both our wild type and mutant varieties of Arabidopsis.

In a damp sterile tray, we proceeded to pipette 20 micro liters of our final concentration on each leaf. The comparing immune response effects between the two different varieties will be very interesting to see!

Leaf Infection

GMO Investigators #2

by Minye Seok

In this lab, we tested for the presence of GMO in food by running gel electrophoresis. First, 5 µl of Orange G loading dye (LD) was added to each sample. Next, 20 µl of the molecular weight ruler was added into the first lane of the 2.5% agarose gel and 20 µl each sample was added into the gel in the order indicated below. Our test foods were Frosted Flakes from Kellogg, General Mills Cocoa Puffs, a donut from Krispy Kreme, and M&Ms.

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The gel was run for 25 minutes at 100 V and the results looked as below. Test food #1 showed positive for the presence of GMO.

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Garden Visit on May 24th (Lab 8)

by Jasmin Revanna and Niharika Loomba

Today we went to the UAB community gardens again! Here, our objective was to find plants that were infected with either a bacterial or fungal disease. To identify plants with bacterial diseases, we looked for soft leaves with white dots surrounded by red or yellow halos. Plants with fungal infections appear to be dried out and decaying towards the edges of the leaves. After about 20 minutes of exploring the gardens and searching for various infected plants, we each gathered a sample of bacteria and fungus infected plants to take back to the lab.

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Pink Group (commonly referred to as Pink Gang) is ready for another day at the gardens!

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Dr. Mukhtar (right) explains the difference between spotting a fungal infection and a bacterial one to Kush Patel (left).

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Found some bacteria-infected plants! Notice the yellow halo around the spots; this is a good way to identify bacteria.

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Here you see a strawberry plant with a fungal infection. Fungal infections are easy to spot because you just have to look for leaves that appear dried out, and some have white fuzzies on them.

 

Once back in the lab, our goal was to isolate the fungal phytopathogens using the samples we gathered in the garden. We cut four small squares from our fungus-infested leaves that contained both healthy and infected tissue and left each square in 10% Clorox solution for a different amount of time. After removing excess Clorox solution from each leaf, we placed the leaf pieces onto a V8 medium plate. Finally, the plates were left covered overnight to allow fungal spores to grow. A similar process will be conducted the next day with bacterial samples.

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Reid Ballard submerges the leaf squares in clorox at different time intervals before plating them, as shown in the next picture.

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After plating, we wait to observe which time interval will allow fungus to grow from the leaf onto the plate.

 

 

GMO Investigators

by Reid Ballard and Lily Birx

In this lab, we brought in food samples to test for the presence of GMOs. First, we weighed out 1 gram of our samples, added 5 mL of distilled water, and ground the mixture until it formed a slurry.

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Photo 1: Weighing out our food samples

We added 25 mL of distilled water and continued to grind our sample until it was smooth enough to pipet in order to make a slurry out of our foods.

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Photo 2: Making food slurries

 

Next, we added 500 uL of InstaGene Matrix and 50 uL of our sample into a screwcap tube and placed in a 93C heat block for 5 minutes.

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Photo 3: Samples in the heat block

 

Then we balanced our sample tubes into a centrifuge and ran them for 5 minutes at max speed.

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Photo 4: centrifuging our samples

We then prepped our samples for PCR, as well as some control samples, adding master mixes to the appropriate samples.

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Photoset 5: Prepping our samples

After prepping our samples, we placed them in the PCR machine and ran PCR.

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Photo 6: PCR machine

Stay tuned for the follow-up experiment, where an electrophoresis gel will be run to determine the presence of GMOs!

The UAB Gardens Adventure

by Carly van Waveren and Jacob Brown

On Wednesday May 16th we had our first field trip!
12We headed to the UAB Community Gardens to get some wild type samples and observe plant pathogens in their natural environment. While we had already infected young Arabidopsis plants with Psm, we had not yet had a chance to observe plant infections in nature. We were told to look through the garden and try to observe the different kinds of plant disease.
Using the knowledge, we gained from Dr. Mukhtars lecture we set out looking for bacterial, viral, and fungal infections. We were told that bacterial infections tended to produce a brown area surrounded by a yellow halo on the plants leaves, while viruses often manifested as white spots or small discolored patches. When placed in solution, bacterial infections would ooze fluid, while viral infections would not. Finally, fungal infections were divided into three categories: biotrophs, necrotrophs, and a hemibiotroph which is a hybrid that can change from a biotroph to a necrotroph.
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Likely a bacterial infection

10Possibly viral due to the white spots and stunted/shriveled appearance
9Likely a fugal necrotrophic or bacteria
8A fungal necrotroph

After collecting samples, we decided to test for viral infections in the plants. A good indicator of a virus in a plant is a yellowed or crinkled leaf. Our TA Katrina passed out ImmunoStrip kits and demonstrated how we should test the leaves for a virus. The kits we used were designed to detect a Potyvirus, one of the largest genus of plant viruses and one that was known for causing significant damage to crops.
14First, we cut a sample of our plant leaf and added it to the ImmunoStrip bag, then we crushed the sample with sharpies and dissolved it into the solution in the bag.
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Afterwards we dipped our test strips into the bags and waited for results. One red line meant the test was negative and two red lines meant it was positive for potyvirus!
5Dr. Mukhtar said that it was very rare for someone to get a positive for a virus and that we would be lucky if even one person found a leaf with the virus. My test came back negative as only one line showed up on the test strip.
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Here is Dr. Mukhtar explaining the different types of infections we could observe while we waited for our test results.
3Amazingly, two people in the group got positives for potyvirus from their ImmunoStrip kits! That’s bad news for the gardeners but good news for us! Dr. Mukhtar told the groups who got positive results to remember which plants they had sampled from so that on our next trip they could collect more samples and bring them back to the lab.
2 After baking in the heat of the gardens we were all ready to head back to the lab and recap what we had learned. Before leaving we snapped a group picture of everyone!
1Visiting the Gardens was a great chance to observe plant pathogens in their natural environment and to see first hand how plants work to defend themselves! We are all looking forward to our next visit where we will collect more samples and attempt to grow cultures from them!

Lab 5 (Pseudomonas Sampling)

by Josh Knapp

Last time we were in the lab we inoculated our Arabidopsis plants with the Pseudomonas syringae bacterium. We had two genetically different Arabidopsis; a wild type columbia plant, and a columbia plant with a mutated NPR1 gene. The mutant plant was more susceptible to pathogenic infection because the NPR1 gene codes for a protein that is crucial to plant immune responses. Knowing this, it was safe to assume that our NPR1 plants would look a little bit worse than our wild type plants after they had sat for a few days, and for the most part they did! Below are the wild type columbia plants and the NPR1 plants.

 

Aside from just looking at the appearance of the infected plants, our goal today was to collect tissue samples from the infected leaves and isolate the syringae colonies within them. We expected to see a greater number of colonies from the NPR1 plants due to their lack of immune response.

To isolate the colonies, we first took two infected leaves from each Arabidopsis plant and punched a hole through them using a standard, classroom hole-puncher. The reason we did this was to make sure that the sample of leaf tissue collected from each plant would be constant, the size of the hole punched, regardless of leaf size. Two “circles” of leaf tissue were added into homogenization tubes that contained Magnesium chloride (MgCl) solution and metallic beads. It is important to mention that we made sure no homogenization tube contained two leaf samples from the same plant, every tube contained two “circles” from two different plants (of the same genotype) in order to ensure that the results would be an average for all of the plants of that genotype. This prevented any one plant from drastically skewing the data if it had unusual results.

 

We then took the samples to be homogenized for six minutes on the highest setting! The homogenizer is basically a machine that just shakes the tubes very rapidly, but because we put the metallic beads inside of the tubes, the beads acted as grinders that broke the leaf tissue down completely.

 

After the samples were well mixed, we added additional MgCl solution to each tube to help dilute the mixtures somewhat.

 

However, we needed to make further dilutions of our bacteria to in order to isolate individual colonies. Our plan was to make a series of six dilutions for each sample tube, and because we had homogenized twelve sample tubes, six containing wild type tissue and six containing NPR1 tissue, we had to prepare 72 wells of MgCl2 solution for the dilutions! Unfortunately supplies were tight, so we couldn’t use a multichannel pipette for this part of the experiment and had to prepare each well the old-fashioned way, by hand.

 

 

 

After the dilutions had been carried out, we transferred 20 μl of the contents from each well onto the large agar plates we had prepared last week. The wild type and NPR1 were kept separate throughout the experiment so we ended up with two agar plates that each contained 36 “drops” of solution, each drop representing the contents of a well from our series of dilutions. Next time we come to lab, we expect to see each sequential drop contain fewer and fewer colonies due to having a lower bacterial concentration. Hopefully, some of the drops will have so few colonies

that we can easily identify and count them, allowing us to see whether more bacteria grew in the NPR1 or wild-type Arabidopsis plants.

UPDATE: Monday, May 21st

When we came back to the lab today we counted the number of colonies that grew on the agar plates. To count the colonies we looked at each of the different “columns” of bacteria, where each sequential “drop” in a column contained a lower bacteria concentration due to our dilutions. We determined which drop had the lowest number of colonies and counted how many colonies had grown in that drop. If there were only one or two colonies in the lowest concentration drop, we would not count that drop and would instead move to the previous drop in the column. We did this because while one or two colonies could have resulted from a very diluted concentration of bacteria, one or two colonies also could have grown randomly, due to some error, so we considered it best not to count any single or double colonies.

On the top is the columbia plate; unfortunately the plate was wet and many of the drops smeared, but it was possible to collect data for five out of the six columns. On the bottom is the NPR1 plate; it has more clearly defined colonies that were easy to count. Additionally, the comparison between these plates highlights how the columbia plants had fewer colonies overall as the dilutions continued.

 

After counting the lowest number of colonies from each column, we put the data for both the wild type and NPR1 agar plates into an excel spreadsheet to compare them. The spreadsheet calculated an average concentration of bacterial growth for each plate, indicating which plant had more bacteria growth overall. As predicted, NPR1 showed higher amounts of bacterial growth, indicating that the mutated plants did have a lower immune response compared to wild type Arabidopsis columbia.