Optimization of Loading Plasmid DNA into Bacterial Outer Membrane Vesicles with Calcium Ions

Clarissa Tan Author

07/27/2021 Added

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My summer research project focused on nonviral oral gene delivery. We bioengineered bacterial OMVs to deliver DNA into our cells in order to treat diseases.

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[00:00:02.182]Hi everyone!
[00:00:03.130]I’m Clarissa, a high school intern with NCMN,
[00:00:05.209]and this is my poster presentation!
[00:00:07.096]This summer, I’ve been in the Biological Systems Engineering Lab
[00:00:10.376]under the supervision of Dr. Pannier,
[00:00:12.386]with tremendous help from my mentors, Kari Heck and Peyton Panovich.
[00:00:15.423]As you can see, our project is the Optimization of Loading
[00:00:18.513]Plasmid DNA into Bacterial Outer-Membrane-Vesicles with Calcium Ions.
[00:00:22.843]This is a specific area of research
[00:00:24.573]under the overall category of gene delivery,
[00:00:26.616]and I'll start by introducing that.
[00:00:29.656]Gene delivery is delivering external material (in our case, DNA)
[00:00:33.419]into our cells to produce a therapeutic effect,
[00:00:35.589]or in other words, to cure diseases.
[00:00:37.629]There are 2 types of gene delivery: viral, and non-viral.
[00:00:40.419]Viral gene delivery is the most effective option,
[00:00:42.515]as viruses have been developing for about a billion years.
[00:00:45.565]However, viral gene delivery can be really dangerous,
[00:00:47.575]which is why we decide to use nonviral gene delivery.
[00:00:50.205]And the only con of this method is that it’s not as effective as viral gene delivery,
[00:00:53.595]which is exactly what our project is working to improve.
[00:00:56.176]Specifically, we use oral nonviral gene delivery,
[00:00:58.795]which means that instead of injection
[00:01:00.445](like getting a vaccination shot)
[00:01:01.755]it would be something more like taking a pill.
[00:01:04.095]There are lots of benefits to oral gene delivery,
[00:01:05.815]a few being that you can administer it by yourself
[00:01:07.415](so you won't need a doctor or nurse),
[00:01:08.915]it’s less painful, it's more efficient,
[00:01:10.825]and the biggest thing is that
[00:01:12.455]oral gene delivery treats diseases
[00:01:13.845]in your intestine and colon.
[00:01:15.505]This is really important because
[00:01:17.125]we don’t get viruses through shots, right,
[00:01:18.755]it’s not like someone stabs you with a needle and you get the flu.
[00:01:21.249]We get viruses through our nose and mouth,
[00:01:23.569]like breathing them in through the air, so on.
[00:01:25.779]Oral gene delivery mimics this by
[00:01:27.339]delivering the DNA through our mouths,
[00:01:28.859]so it is the most effective way of fighting the disease.
[00:01:31.789]However, oral DNA delivery is difficult because
[00:01:34.169]many of the delivery vehicles are disintegrated by acid in our stomach,
[00:01:37.879]thus failing to protect the DNA
[00:01:39.589]that’s supposed to enter our cells.
[00:01:40.955]So the next thing that we needed to focus on in this project
[00:01:42.875]was finding the ideal delivery vehicle,
[00:01:44.675]which we would load the DNA into.
[00:01:47.775]We decided to use OMVs, or outer-membrane-vesicles.
[00:01:50.865](and there's a diagram of OMVs here to the right)
[00:01:53.885]OMVs come from the e coli strain of bacteria,
[00:01:55.769]they’re commonly used as communication systems.
[00:01:58.207]The reasons why OMVs are good for our project are
[00:02:00.507](a), they can protect the DNA from
[00:02:02.277]being disintegrated by stomach acid,
[00:02:04.161]and (b) they still retain their activity
[00:02:06.171]while passing through the GI (gastrointestinal) tract.
[00:02:09.707]So, as mentioned before,
[00:02:10.857]nonviral gene delivery is not as efficient as viral gene delivery.
[00:02:13.621]Thus, our objective is to maximize this efficiency
[00:02:16.131](how much DNA gets loaded into our cells).
[00:02:18.239]In order to do this,
[00:02:19.332]we hypothesize that adding Calcium ions would speed up the process.
[00:02:23.608]Okay, so now onto the process of
[00:02:24.678]how we actually loaded and measured these OMVs.
[00:02:26.808]First, we had to grow the e coli bacteria in broth,
[00:02:29.115]run it through a filter, and then
[00:02:30.445]centrifuge the tubes to separate the OMVs from the bacteria.
[00:02:33.114]Next, we ran a BCA assay
[00:02:34.994]to measure the protein content in our OMVs
[00:02:36.884](and this number will be important later on),
[00:02:39.184]and we also took a picture of the OMVs.
[00:02:41.124]Next, we did the loading assay,
[00:02:42.164]in which we combined Calcium, DNA, and OMVs
[00:02:44.988]in various ratios, and electroporated them.
[00:02:47.748]We also used the protein calculations
[00:02:49.558]from earlier in order to do these calculations.
[00:02:53.141]Finally, we added the Hoescht fluorescent dye
[00:02:55.561]and ran it through the plate reader to
[00:02:56.581]measure the amount of DNA in each sample.
[00:02:58.341]To do our final calculations for loading efficiency,
[00:03:00.731]we just used the formula on the screen here.
[00:03:02.715]So we did the DNA measured in sample
[00:03:04.747]and we subtracted from that,
[00:03:06.327]the DNA in the OMV control, and then
[00:03:08.217]we divided that by the intial amount
[00:03:09.498]of DNA we used in the experiment,
[00:03:11.408]and then multiplied by 100 to get the percentage.
[00:03:14.368]And then here are the results!
[00:03:15.768]You can see in the 1st graph that
[00:03:17.112]adding Calcium significantly increased loading efficiency,
[00:03:19.532]and to get even more specific,
[00:03:20.942]the 2nd graph shows that a 1:4 ratio of DNA:OMV (with Calcium)
[00:03:24.752]yields the highest loading efficiency.
[00:03:26.769]The next 2 graphs show transfection efficiency,
[00:03:28.699]which is how much DNA was actually retained
[00:03:30.969]when the OMV-loaded nanocarriers were put into the cells.
[00:03:35.013]So in conclusion, our hypothesis was correct,
[00:03:37.243]Calcium does increase loading and transfection efficiencies.
[00:03:40.063]We’ve also shown that OMV can actually be loaded with DNA,
[00:03:42.986]and that electroporation voltage helps with the loading process.
[00:03:45.931]For future directions, in the short-term,
[00:03:47.711]we would like to try more
[00:03:48.871]positively-charged elements similar to Calcium,
[00:03:51.340]as well as more variations of the DNA:OMV ratio.
[00:03:53.978]In the (I guess) medium-term future,
[00:03:56.096]we want to simulate these DNA-OMV nanocarriers
[00:03:59.228]passing through the GI tract in the lab,
[00:04:01.053]and we'll probably also test it on the GI tracts of mice.
[00:04:03.553]And in the long-term,
[00:04:04.672]we want this research to make oral nonviral gene delivery medicines,
[00:04:07.912](such as vaccine pills)
[00:04:08.752]to be widely available to the public,
[00:04:10.512]so that maybe one day, you can give yourself a vaccine at home.
[00:04:13.558]Finally, I would like to give a huge thanks
[00:04:15.898]to everyone in Dr. Pannier’s lab for all of their help this summer.
[00:04:18.448]I especially want to thank Kari and Peyton
[00:04:20.018]for mentoring me every day on this project.
[00:04:21.796]Finally, I want to thank NCMN
[00:04:23.566]for making this internship possible,
[00:04:25.106]the J.A. Woollam company, the NSF, and other sponsors. Thank you :)))

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Optimization of Loading Plasmid DNA into Bacterial Outer Membrane Vesicles with Calcium Ions

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