Improving Bioplastic Production in Rhodopseudomonas palustris CGA009

Dylan Hoppner Author

04/04/2021 Added

13 Plays

Description

Searchable Transcript

Search:

[00:00:04.290]Hello, my name is Dylan Hoppner
[00:00:05.797]and I am a member of the Systems and Synthetic Biology Lab
[00:00:08.390]here on the campus of University of Nebraska Lincoln.
[00:00:11.790]Today I'll be presenting to you my research project,
[00:00:15.160]Improving Bioplastic Production
[00:00:16.660]in Rhodopseudomonas palustris CGA009.
[00:00:20.320]I would like to give a shout out to Dr. Cheryl Immethun
[00:00:23.960]for her help with my project.
[00:00:27.000]Most of the work demonstrated here today
[00:00:29.180]would not have been possible without her.
[00:00:34.920]So to start off,
[00:00:37.540]why am I choosing Rhodopseudomonas palustris
[00:00:40.257]and what am I trying to accomplish?
[00:00:43.610]So here's a picture of Rhodopseudomonas palustris.
[00:00:46.090]I will shorten that to R. palustris
[00:00:48.280]for the remainder of this presentation.
[00:00:51.060]I chose Rhodopseudomonas palustris
[00:00:54.160]because it is metabolically versatile
[00:00:55.690]which means it can utilize different aspects
[00:00:57.570]of metabolizing products into energy.
[00:01:01.760]This is very flexible for different aspects of the industry
[00:01:05.620]but R. palustris specifically is successful
[00:01:09.220]at converting ethanol waste product namely lignin
[00:01:12.970]into valuable bioplastics.
[00:01:17.330]Normally this waste product is burned
[00:01:20.780]for energy or it's fed to cattle.
[00:01:23.200]And instead what we would do is we would take
[00:01:25.640]R. palustris and modify it.
[00:01:27.380]So it eats this
[00:01:31.760]lignin waste product and converts
[00:01:33.120]it into something valuable which reduces
[00:01:35.680]the amount of pollution in the atmosphere
[00:01:37.450]by reducing the amount of greenhouse gases
[00:01:39.310]that you make when you burn something.
[00:01:41.870]But it also creates something valuable because as engineers
[00:01:45.330]we like to squeeze every little bit of value we can get
[00:01:48.650]out of our raw materials in this case,
[00:01:51.080]which that would be corn.
[00:01:54.910]Moving on, I propose two different research questions.
[00:01:58.780]First being, does increasing the propanoyl-CoA pool
[00:02:01.570]increase the addition of R-3-Hydroxyvaleryl-CoA
[00:02:05.643]into PHB or PHBV?
[00:02:09.580]So palustris actually already naturally
[00:02:12.000]makes this bioplastic PHB and PHV.
[00:02:15.910]What it does is utilizes chemicals like propanoyl-CoA
[00:02:19.910]and acetyl-CoA and converts them
[00:02:21.430]into phaA and then converts that into phaB
[00:02:26.030]and then finally converts that
[00:02:27.110]into our valuable bioplastic phaC.
[00:02:31.930]So what we wanna do is we wanna increase the amount
[00:02:35.360]of available propanoyl-CoA, that palustris makes
[00:02:39.590]so that it will make more valuable bioplastic.
[00:02:47.440]PHB and PHBV is valuable because as I've stated before
[00:02:51.640]it's biodegradable, it has many different single use
[00:02:54.810]and medical applications but PHBV is more desirable
[00:02:59.230]because it's easier to process and PHB
[00:03:03.080]its physical properties make it more flexible
[00:03:06.340]for industry standards and has a lot more uses than PHB.
[00:03:13.650]All right, research question is, does knocking down
[00:03:17.150]reactions that consume propanoyl-CoA
[00:03:19.260]increase the available metabolite?
[00:03:23.110]Here's a graphic demonstrating some of the genes
[00:03:26.130]that R. palustris has that makes and uses propanoyl-CoA.
[00:03:32.120]The genes we are targeting would be RPA 3175,
[00:03:35.894]RPA 2394 and RPA 4567.
[00:03:40.350]That is the star genes down here.
[00:03:42.950]As you can see, they consume propanoyl-CoA
[00:03:45.420]and convert it into other products.
[00:03:47.570]And so we wanna know if repressing or knocking down
[00:03:51.760]these genes increases the amount of available propanoyl-CoA
[00:03:55.180]that palustris has to make the bioplastic that weak desire.
[00:03:59.770]Unfortunately RPA 2394, this guy right here
[00:04:04.380]has no documentation and any scientific database.
[00:04:09.200]So we can not successfully make genetic sequences
[00:04:12.950]in order to repress this gene in palustris.
[00:04:15.520]So we only focused on the other two,
[00:04:17.920]3174 and 4567
[00:04:20.080]in our experiments.
[00:04:24.530]So how will we go about increasing the amount
[00:04:26.620]of propanoyl-CoA or utilizing propanoyl-CoA
[00:04:29.040]to make our valuable bioplastics?
[00:04:31.700]So we will be using the CRISPRi system
[00:04:33.900]and there is a little graphic showing what it does
[00:04:36.680]but to explain it, what we do is we design
[00:04:39.440]some small guide RNA.
[00:04:41.110]And what that small guide RNA does is it acts
[00:04:44.100]as a little flag and it tells the dCas9 protein where to go.
[00:04:48.480]And the dCas9 protein goes and sits on top of the gene
[00:04:52.670]that we want to repress and it just blocks gene expression.
[00:04:56.490]So it stops the gene from being expressed in R. palustris.
[00:05:01.360]How do we design the small guide RNA?
[00:05:03.450]Well what we do is we utilize a internet scientific database
[00:05:06.560]CHOPCHOP and what we do is we implement our gene of interest
[00:05:10.420]target site right there
[00:05:12.270]and our organism Rhodopseudomonas palustris?
[00:05:15.210]What system we use?
[00:05:16.270]CRISPRi and what we want it to do
[00:05:18.690]for repression or knockdown.
[00:05:21.000]Using CHOPCHOP it actually gives us an accurate
[00:05:25.100]sgRNA sequence and it tells us the efficacy of the sequence.
[00:05:29.020]And it also provides valuable information
[00:05:30.510]such as the GC-content of the sequence
[00:05:34.100]that we use when we design our DNA sequences.
[00:05:38.940]Now, once we have this small guide RNA
[00:05:40.740]what do we do with it?
[00:05:42.010]Well, in this graphic shown on the slide
[00:05:44.220]I created a workflow map and so you see
[00:05:46.900]we start with CHOPCHOP to choose and design small guide RNA.
[00:05:50.770]What we then do with that small guide RNA
[00:05:52.650]is we use it and put it in a plasmid.
[00:05:56.070]Now building this plasmids is a quite long
[00:05:59.180]and complicated process.
[00:06:00.360]You can read some of the bulleted points
[00:06:01.860]and see that there's a lot of stuff involved
[00:06:04.780]with building a plasmid but it takes several weeks
[00:06:10.070]to build and complete a plasmid.
[00:06:11.740]But once you have the finished plasmid
[00:06:14.980]what you do is you use hot fusion
[00:06:17.310]to ligate everything together for your finished plasmid.
[00:06:21.550]And then what you do is you take the finished plasmid
[00:06:24.080]and you transform it into E. coli using electroporation
[00:06:26.860]which is you send a lot of electricity through E. coli
[00:06:30.427]and it creates small little holes.
[00:06:32.350]And then the DNA floats into those holes
[00:06:34.480]and implements itself into E. coli genom.
[00:06:37.780]Once you grow E. coli under a antibiotic
[00:06:42.280]creating selection pressure, you mini prep the plasmid,
[00:06:45.160]you PCR the plasmid and then you gel visualize
[00:06:47.920]making sure that the plasmid is the right size.
[00:06:51.960]And it gives you a rough idea of how correct it is.
[00:06:54.890]And then once you have the gel visualization
[00:06:57.060]what you do is you send the plasmid off
[00:06:59.980]to be sequence verified.
[00:07:01.310]And once it sequence verified we can transform
[00:07:03.940]it into R. palustris.
[00:07:05.660]And then after that you grow R. palustris
[00:07:07.530]with the plasmid in it, and you do the exact same step
[00:07:11.380]the mini prep, the PCR, the gel visualization
[00:07:13.770]and then the sending and off to sequencing.
[00:07:16.600]You send it off the sequencing to make sure
[00:07:18.040]that palustris hasn't mutated the plasmid in any way
[00:07:21.810]so that you know, that the genes
[00:07:23.200]are being knocked down correctly, and yeah.
[00:07:30.480]Once you have a sequence verified plasmid
[00:07:32.210]what you do is we will control wild type palustris
[00:07:36.780]that means with no additional genetic modification
[00:07:40.020]and our CRISPRi modified palustris.
[00:07:42.690]And we will measure the intracellular propanoyl-CoA
[00:07:45.200]and PHBV content of each group
[00:07:47.860]and see if there's been a change, increase or decrease
[00:07:50.810]and confirming whether our plasmid
[00:07:53.370]is functional and working.
[00:07:56.030]Currently in the workflow map
[00:07:57.490]we sit right here at the sequence verified plasmid stage.
[00:08:01.030]We have grown palustris with a plasmid
[00:08:04.020]and it demonstrates antibiotic resistance.
[00:08:07.230]So we know that some gene expression at the plasmid
[00:08:10.290]is working properly and we just need to sequence verify it.
[00:08:15.040]So once we've completed all that and we've measured
[00:08:17.460]the difference between controlled palustris
[00:08:20.940]and our CRISPRi modified palustris
[00:08:23.250]the next steps of the project are actually
[00:08:26.320]streamlining the design of our plasmid.
[00:08:29.520]So before there was several weeks involved worth of work
[00:08:34.200]of cutting up previous backbones
[00:08:36.270]ligating them together, ligating in our hot fusion,
[00:08:40.330]using hot fusion to put it in our small guide RNA.
[00:08:43.010]And we'll streamline that into one
[00:08:45.280]universal simple backbone
[00:08:47.810]and then you implement your different small guide RNA
[00:08:51.040]into that for each different experiments.
[00:08:54.040]And then eventually we utilize that background
[00:08:56.200]to implement several small guide RNA in at the same time.
[00:08:59.270]So we can repress several genes at once
[00:09:02.090]using the simple backbone.
[00:09:06.520]I would like to thank everyone
[00:09:07.750]in the Systems and Synthetic Biology Lab.
[00:09:09.760]At one point or another someone helps me with my project
[00:09:12.160]and I'm very grateful for that
[00:09:13.870]but I would like to give a big shout out to Dr. Rajib Saha
[00:09:17.310]and Dr. Cheryl Immethun who without their help
[00:09:20.880]I would be nowhere near the product that I have today.
[00:09:26.800]And I would also like to thank just, you know
[00:09:28.250]Clark for helping me get set up through UCARE
[00:09:30.660]and go through the process of applying and getting accepted.
[00:09:34.300]And I'd also like to give a thanks to UCARE itself
[00:09:37.270]for funding my project and giving me the opportunity to work
[00:09:40.090]in this lab with all these wonderful people.
[00:09:42.800]Thank you for listening to my presentation.

The screen size you are trying to search captions on is too small!

You can always jump over to MediaHub and check it out there.

Comments

0 Comments

Improving Bioplastic Production in Rhodopseudomonas palustris CGA009

Description

Searchable Transcript

Comments icon comment

Related Channels

Comments