iGEM 2023 — Team austin-utexas

'use client';
import Figures from '../components/Figures';
import Footer from '../components/Footer';
import Navigation from '../components/Navigation';
import Head from 'next/head';

const notebook = () => {
return (
<>
<Navigation name="UT Austin iGEM 2023"/>

<Head>
    <title>Notebook</title>
    <link rel="shortcut icon" href="https://static.igem.wiki/teams/4579/wiki/logo-removebg-preview.png" />
</Head>

<h1 className="animate-fade-up">Notebook</h1>
<h4></h4>
<section>May 2023</section>
<h3>
<ul className = "list-inside list-image-bacteriaBullet">
<li> <></> <a href ="https://static.igem.wiki/teams/4579/wiki/week-5-8-12-benchling.pdf" className="underline"> 5/8-5/12 </a> </li>
<li> <></> <a href ="https://static.igem.wiki/teams/4579/wiki/week-5-15-20-benchling.pdf" className="underline"> 5/15-5/20 </a> </li>
<li> <></> <a href ="https://static.igem.wiki/teams/4579/wiki/benchling-week-5-22-26.pdf" className="underline"> 5/22-5/26 </a> </li>
<li> <></> <a href ="https://static.igem.wiki/teams/4579/wiki/week-5-23-26-benchling.pdf" className="underline"> 5/23-5/26 </a></li>
</ul></h3>
<section>June 2023</section>
<h3>
<ul className = "list-inside list-image-bacteriaBullet">
<li> <></> <a href ="https://static.igem.wiki/teams/4579/wiki/week-5-29-6-4-benchling.pdf" className=" underline"> 5/29-6/4 </a> </li>
<li> <></> <a href ="https://static.igem.wiki/teams/4579/wiki/week-6-5-6-9-benchling.pdf" className="underline"> 6/5-6/9 </a> </li>
<li> <></> <a href ="https://static.igem.wiki/teams/4579/wiki/week-6-12-6-16-benchling.pdf" className=" underline"> 6/12-6/16 </a> </li>
<li> <></> <a href ="https://static.igem.wiki/teams/4579/wiki/week-6-19-6-23-benchling.pdf" className="underline"> 6/19-6/23 </a></li>
<li> <></> <a href ="https://static.igem.wiki/teams/4579/wiki/week-6-26-6-30-benchling.pdf" className="underline"> 6/26-6/30 </a> </li> </ul>
</h3>

<section>July 2023</section>
<h3>
<ul className = "list-inside list-image-bacteriaBullet">
<li> <></> <a href ="https://static.igem.wiki/teams/4579/wiki/week-7-3-7-7-benchling.pdf" className="underline"> 7/3-7/7 </a> </li>
<li> <></> <a href ="https://static.igem.wiki/teams/4579/wiki/week-7-10-7-14-benchling.pdf" className="underline"> 7/10-7/14 </a> </li>
<li> <></> <a href ="https://static.igem.wiki/teams/4579/wiki/week-7-17-7-21-benchling.pdf" className="underline"> 7/17-7/21 </a></li>
<li> <></> <a href ="https://static.igem.wiki/teams/4579/wiki/week-7-24-7-28-benchling.pdf" className="underline"> 7/24-7/28</a></li></ul>
</h3>

<section>August 2023</section>
<h3>
<ul className = "list-inside list-image-bacteriaBullet">
<li> <></> <a href ="https://static.igem.wiki/teams/4579/wiki/week-7-31-8-4-benchling.pdf" className="underline"> 7/31-8/4 </a> </li>
<li> <></> <a href ="https://static.igem.wiki/teams/4579/wiki/week-8-21-8-25-benchling.pdf" className="underline"> 8/21-8/25</a></li> </ul> </h3>

<section>September 2023</section>
<h3>
<ul className = "list-inside list-image-bacteriaBullet">
<li> <></> <a href ="https://static.igem.wiki/teams/4579/wiki/week-8-28-9-1-benchling.pdf" className="underline"> 8/28-9/1 </a> </li>
<li> <></> <a href ="https://static.igem.wiki/teams/4579/wiki/week-9-3-9-9-benchling.pdf" className="underline"> 9/3-9/9 </a></li> </ul> </h3>

<h5></h5>

<Footer Year='2023'/>
</>
)
}

export default notebook

'use client';
import { AccordionPanel } from 'flowbite-react/lib/esm/components/Accordion/AccordionPanel';
import Cards from '../components/Cards';
import Figures from '../components/Figures';
import AccordianFigure from '../components/AccordianFigure';
import Footer from '../components/Footer';
import Navigation from '../components/Navigation';
import Sources from '../components/Sources';
import { Accordion } from 'flowbite-react';
import Head from 'next/head';

const engineering = () => {
return (
<>
<Navigation name="UT Austin iGEM 2023"></Navigation>
<Head>
    <title>Engineering Success</title>
    <link rel="shortcut icon" href="https://static.igem.wiki/teams/4579/wiki/logo-removebg-preview.png" />
</Head>
<h1 className='animate-fade-up'>Engineering Success</h1>
<h4 className = 'pb-10'></h4>
<h3 className='flex justify-center'></h3>
<h3 className='flex justify-center'><img className='w-1/3 h-auto rounded-full' src='https://static.igem.wiki/teams/4579/wiki/overall-dbtl-1.png'/></h3>
<section>Introduction</section>
<h3>To develop a system that would allow us to combat plant pathogens, our team had <a className='font-bold'>three primary objectives</a> (Figure 1):
<ul className='list-inside list-image-bacteriaBullet ml-6 py-4'>
  <li><a href = '#Objective1' className='font-bold underline hover:no-underline text-DMagenta'>Objective 1: Redesigning an Existing Microcin Expression Plasmid to be More Modular</a></li>
  <li><a href = '#Objective2' className='font-bold underline hover:no-underline text-DMagenta'>Objective 2: Predicting Novel Microcins</a></li>
  <li><a href = '#Objective3' className='font-bold underline hover:no-underline text-DMagenta'>Objective 3: Adapting Optimized Inducible Promoters for Our System</a></li>
  </ul> Pursuing and achieving these objectives provided the framework for implementing our system in our bioassay procedures and pipeline. </h3>
<h3 className = 'flex justify-center pt-4 pb-2'>
        <img src='https://static.igem.wiki/teams/4579/wiki/engineering-objectives-overview-4.png' className=" mx-15 w-full h-auto rounded-xl border-solid border-BGreen border-2 hover:drop-shadow-lg" />
        </h3>
        <h3 className = 'text-Avocado italic text-center pt-2 pb-4'>Figure 1. Overview of the three objectives of our engineering process. Created with BioRender.com.</h3>
<h3>Our first objective was to <a className='font-bold'>redesign an existing microcin expression plasmid to be a more modular system</a> (Kim et al. 2023). This enabled us to easily swap in different microcins and expression regulators without needing to alter the rest our genetic circuit. Building off of the foundation set by our redesigned system, our second objective was to <a className='font-bold'>predict novel microcins that could potentially target our pathogens of interest</a>. In tandem with this second objective, we also pursued our third objective, in which we <a className='font-bold'>adapted optimized inducible promoters for use in our system</a> (Meyer et al., 2019). </h3>
<h3>
For each objective, we underwent at least one iteration of the <a className='font-bold'>Design, Build, Test, and Learn (DBTL) cycle</a>. In general, we relied on <a className='font-bold'>Golden Gate Assembly (GGA)</a> (Engler et al. 2008, Engler et al. 2009) to build the specific constructs used for each objective (Figure 2). To examine whether an objective was accomplished, we often conducted <a className='font-bold'>zone of inhibition (ZOI) assays</a> that allowed us to observe whether a microcin’s behavior aligned with our expectations (Figure 2). When the results of our testing procedures suggested the presence of problems, we reexamined relevant literature, our initial designs, and/or our testing methods to inform the changes we needed to make. </h3>
<h3 className = 'flex justify-center pt-4 pb-2'>
        <img src='https://static.igem.wiki/teams/4579/wiki/gga-5.png' className=" mx-15 w-full h-auto rounded-xl border-solid border-BGreen border-2 hover:drop-shadow-lg" />
        </h3>
        <h3 className = 'text-Avocado italic text-center pt-2 pb-4'>Figure 2. Overview of the Golden Gate Assembly (GGA) cloning process used to build our DNA constructs and the ZOI assay procedure used to test them. Created with BioRender.com.</h3><div id="Objective1"></div><h5></h5>

<h4></h4>
<section>Objective 1: Redesigning an Existing Microcin Expression Plasmid to be More Modular </section>
<h3>We improved the modularity of the existing microcin V (MccV) expression plasmid found within a two-plasmid MccV secretion system (Kim et al. 2023). To do so, we created <a className='font-bold'>a generalized assembly scheme</a> for how the genetic components of microcin expression are put together in a transcriptional unit. We then demonstrated that MccV was still effective in that scheme. See the <a className='font-bold text-DMagenta underline hover:no-underline' href='https://2023.igem.wiki/austin-utexas/description'>Project Description Page</a> for more on microcin expression and the original MccV system. </h3>
<h3><Accordion collapseAll>
<Accordion.Panel >
<Accordion.Title className='text-white bg-DMagenta focus:bg-LGreen focus:ring-DMagenta focus:ring-2 hover:bg-LGreen hover:ring-white hover:ring-4'><p className='text-darkWhite'>
Objective 1 Engineering Cycle <span className = 'flex justify-right text-sm text-white underline'> Click to Expand</span> </p>
</Accordion.Title>
<Accordion.Content className='bg-DMagenta text-darkWhite'>
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<Accordion.Title className='text-white bg-accordion1 focus:bg-LGreen focus:ring-DMagenta focus:ring-2 hover:bg-LGreen hover:ring-white hover:ring-4 pb-2 text-xs'><p className='text-darkWhite '>Design <span className = 'text-right text-sm text-white underline align-right'> Click to Expand</span></p></Accordion.Title>
<Accordion.Content className='bg-accordion1 text-darkWhite'>
<p>We defined the components of microcin expression using the GGA part type categorization described in Leonard et al. 2018 (Figure 3). This allows genetic components of the same part type, such as the microcin and the immunity protein, to be easily interchanged with one another. We <a className='font-bold'>made the CvaC15 signal peptide its own part to enable any microcin to be fused to the signal</a> and recognized for secretion. See the <a className='font-bold text-lavender underline hover:no-underline' href='https://2023.igem.wiki/austin-utexas/parts'>Parts Page</a> for more details on how we used these overhangs (Leonard et al. 2018) to define and arrange the different parts in our modular microcin expression system. </p>
<br></br>
<AccordianFigure
imageSource='https://static.igem.wiki/teams/4579/wiki/our-system-vs-davies.png'
figureLegend='Figure 3. The original MccV expression plasmid versus the general assembly schema for our modular microcin expression plasmid. Created with BioRender.com.'/>
<p>
We designed primers to amplify the <a className = 'italic'>E. coli</a> genes <a className = 'italic'>cvaC</a> (MccV) and <a className = 'italic'>cvi</a> (immunity protein for MccV) from the original MccV expression plasmid. Because the nucleotide sequence for the CvaC15 signal peptide was too short to design primers for, we designed a pair of complementary oligonucleotides that could be annealed together to form the part. For each part, we ensured that the primers/oligonucleotides added BsmBI and BsaI restriction sites with overhangs that enable the part to fit into our assembly scheme. When <a className = 'italic'>cvaC</a>, <a className = 'italic'>cvi</a>, and CvaC15 are assembled according to this scheme, they form a full transcriptional unit for expressing MccV.</p>
</Accordion.Content>
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<Accordion.Title className='text-white bg-accordion2 focus:bg-LGreen focus:ring-DMagenta focus:ring-2 hover:bg-LGreen hover:ring-white hover:ring-4 pb-2 text-xs'><p className='text-darkWhite'>Build <span className = 'text-right text-sm text-white underline'> Click to Expand</span></p></Accordion.Title>
<Accordion.Content className='bg-accordion2 text-darkWhite'><br></br>
<p>Using the primers we designed, we performed Polymerase Chain Reactions (PCR) to amplify <a className = 'italic'>cvaC</a> and <a className = 'italic'>cvi</a> as parts adapted to our GGA standard. To obtain our CvaC15 part, we used a slow-cooling thermocycler protocol to anneal the two complementary single-stranded oligonucleotides we designed together. Once we confirmed that the sizes of each of our parts were correct using gel electrophoresis (Figure 4), we purified them using the Zymo DNA Clean and Concentrate Kit.</p><br></br>
<p>We then created two basic parts, one consisting of both <a className = 'italic'>cvaC</a> and <a className = 'italic'>cvi</a> and the other consisting of CvaC15, by using GGA to clone the parts into a high-copy GFP-dropout entry vector (Leonard et al., 2018) via BsmBI digestion-ligation. Once each basic part was sequence confirmed, we used GGA to assemble these two parts together, along with BTK constitutive promoter (CP25) and terminator (<a className = 'italic'>rpoC</a>) parts, into a BTK low-copy backbone via BsaI digestion-ligation (Leonard et al., 2018). The resulting assembly formed a constitutive MccV expression plasmid. Using whole plasmid sequencing, we confirmed that the plasmid we designed <a className = 'italic'>in silico</a> had been assembled correctly (Figure 4).
</p><br></br>
<AccordianFigure
imageSource='https://static.igem.wiki/teams/4579/wiki/phase-1-sequencing-3.png'
figureLegend='Figure 4. Successful sequencing results for our MccV expression plasmid. The construct that we designed in silico is on top and the sequencing result is on bottom. We used Plasmidsaurus which uses Oxford Nanopore sequencing technology which can have errors in reading stretches of repeated nucleotides. This can result in the appearance of mismatches when aligning the sequences, so we manually examined mismatched regions and confirmed that they were within the backbone and not within the transcriptional unit.'/><br></br>
<p>We then electroporated strains of <a className='italic'>E. coli</a> DH5α and the agricultural biocontrol <a className='italic'>Pantoea vagans</a> C9-1 with our redesigned MccV expression system along with the <a className='font-bold'>secretion system plasmid of the original system (pSK01)</a>.</p><br></br>
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<Accordion.Title className='text-white bg-accordion3 focus:bg-LGreen focus:ring-DMagenta focus:ring-2 hover:bg-LGreen hover:ring-white hover:ring-4 pb-2 text-xs'><p className='text-darkWhite'>Test <span className = 'text-right text-sm text-white underline'> Click to Expand</span></p></Accordion.Title>
<Accordion.Content className='bg-accordion3 text-darkWhite'>
<p className='underline'>Testing MccV in Our Modular Microcin Expression System</p>
<p>To test if the redesigned MccV microcin expression plasmid functions as intended, we performed Zone of Inhibition (ZOI) assays (Figure 5). As detailed on the <a className='font-bold text-lavender underline hover:no-underline' href='https://2023.igem.wiki/austin-utexas/experiments'>Experiments Page</a> , we spotted 10 uL of a concentrated overnight culture of each predator strain onto LB agar plates topped with a layer of soft LB agar containing the prey strain. For MccV, the prey strain was <a className='italic'>E. coli</a> W3110. The predator strains were either <a className='italic'>E. coli</a> DH5α or <a className='italic'>P. vagans</a>. In this experiment, each predator is expressing MccV while the prey is susceptible to this microcin. In Figure 5, the first plate, spot 1 contains our redesigned modular MccV microcin expression plasmid. Spot 2 contains the original MccV expression plasmid from Kim et al. </p><br></br>
<AccordianFigure
imageSource='https://static.igem.wiki/teams/4579/wiki/ecoli-and-1595-mccv-recovered-3.png'
figureLegend='Figure 5. Result of ZOI assays with E. coli DH5α (left) and P. vagans C9-1 (right) strains. Left: MccV expression plasmid (1), original MccV expression plasmid (2), MccV expression plasmid with pSK01 (3, 4), neither plasmid (5, 6). Right: MccV expression plasmid (1), neither plasmid (2). Created with BioRender.com.'
/><br></br>
<p>After overnight incubation, we observed that strains containing our redesigned MccV modular microcin expression plasmid displayed visible zones of inhibition.</p><br></br>
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<Accordion.Title className='text-white bg-accordion4 focus:bg-LGreen focus:ring-DMagenta focus:ring-2 hover:bg-LGreen hover:ring-white hover:ring-4 pb-2 text-xs'><p className='text-darkWhite'>Learn <span className = 'text-right text-sm text-white underline'> Click to Expand</span></p></Accordion.Title>
<Accordion.Content className='bg-accordion4 text-darkWhite'>
<p>The production of a ZOI by both strains containing our redesigned MccV modular microcin expression plasmid indicated that <a className='font-bold'>MccV, when used in our system, was still expressed, secreted, and effective against its target</a>. This demonstrated that adapting the components of microcin expression to the GGA standard and assembly scheme we created still allows for proper microcin expression and secretion. As such, we learned that <a className='font-bold'>our modular microcin expression system is functional</a>.</p><br></br>
<p>Additionally, because <a className='font-bold'>our system was shown to function in the </a><a className='italic font-bold'>P. vagans</a><a className='font-bold'> C9-1 strain, we confirmed that our system works outside of </a><a className='italic font-bold'>E. coli</a><a className='font-bold'> and that</a> <a className='italic font-bold'>P. vagans</a><a className='font-bold'> C9-1 is a suitable chassis for our bioassays</a>. Thus, these results gave us evidence that <a className='font-bold'>our system was successfully constructed and functions as intended</a>.</p><br></br>
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</Accordion></h3><div id="Objective2"></div><h5></h5>

<h4></h4>
<section>Objective 2: Predicting Novel Microcins</section>
<h3>After confirming that our redesigned modular microcin expression plasmid worked, we applied our GGA standard to the microcins predicted from <a className='italic'>cinful</a> (Cole et al., 2022) and used them to produce microcin expression plasmids according to our assembly schema. See the <a className='font-bold text-DMagenta underline hover:no-underline' href='https://2023.igem.wiki/austin-utexas/description'>Project Description Page</a> for more on <a className='italic'>cinful</a>.</h3>
<h3> Initially, we predicted microcins that could target the following <a className = 'font-bold'>4 strains of pathogenic Pantoea:</a> <a className = 'italic'>P. agglomerans</a> PNG 92-11, <a className = 'italic'>P. allii</a> PNA 200-10, <a className = 'italic'>P. ananatis</a> LMG 2665, and <a className = 'italic'>P. ananatis</a> PNA 97-1R. Later in our project, we also predicted microcins for strains of <a className = 'font-bold'>pathogenic </a><a className = 'font-bold italic'>Erwinia amylovora</a> <a className = 'font-bold'>and</a> <a className = 'font-bold italic'>Xanthomonas</a>. <a className = 'font-bold'> After several repetitions of the DBTL cycle</a>, we were able to <a className = 'font-bold'>demonstrate promising antimicrobial activity against one of our target strains</a>.
</h3>
<h3><Accordion collapseAll>
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<Accordion.Title className='text-white bg-DMagenta focus:bg-LGreen focus:ring-DMagenta focus:ring-2 hover:bg-LGreen hover:ring-white hover:ring-4'><p className='text-darkWhite'>
Initial Engineering Cycle <span className = 'flex justify-right text-sm text-white underline'> Click to Expand</span> </p>
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<Accordion.Content className='bg-accordion1 text-darkWhite'>
<p>In the hopes of maximizing the likelihood that the <a className='italic'>Pantoea</a> microcins we selected for further study would be effective, we <a className='font-bold'>filtered</a> <a className='font-bold italic'>cinful’s</a> <a className='font-bold'>output</a> for microcins that met the following criteria:  </p><br></br>
<ul className = "list-inside mx-2 list-image-bacteriaBullet">
<li> <></> A <a className='font-bold'>signal peptide sequence was identified</a> at the beginning of the microcin (“signalMatch = TRUE”)</li>
<li> <></> The HMMER biosequence analysis program used by <a className = 'italic'>cinful</a> found that the microcin amino acid sequence had a <a className='font-bold'>significant degree of similarity</a> with any of the 10 known microcins (“hmmerHit = TRUE”) </li>
</ul><br></br>
<p>The filtered microcins were then aligned using MUSCLE (Madeira et al. 2022) to ensure that the microcins we selected were not duplicates of one another. The alignment indicated that there were <a className = 'font-bold'>six unique microcin sequences</a>, so we chose to work with all six (Figure 6).</p><br></br>
<AccordianFigure
imageSource='https://static.igem.wiki/teams/4579/wiki/microcin-alignment-tree.png'
figureLegend='Figure 6. MUSCLE alignment of the amino acid sequences of the filtered Pantoea microcins predicted by cinful, with the strain of origin of each microcin indicated on the left. The 90 filtered microcins were clustered into 6 families of unique sequences.'/>
<p>To adapt these microcins to our assembly scheme, we removed the native 15-18 AA-long signal peptide sequence. This signal was found at the beginning of each microcin AA sequence and often ended in a GG, GA, or GS (Parker & Davies 2022). Removing the 15-18 AA-long signal peptide sequence allowed the microcin to fuse with our system’s CvaC15 signal peptide. We then obtained the original nucleotide sequence for each microcin by using information in <a className = 'italic'>cinful’s</a> output to locate the microcin sequence in its original genome. We then added BsmBI and BsaI restriction sites that produced the correct overhangs according to our GGA standard (see the <a className='text-bold underline hover:no-underline text-lavender' href = 'https://2023.igem.wiki/austin-utexas/parts'>Parts Page</a> for more details). We ordered the redesigned microcins as gBlocks from Integrated DNA Technologies (IDT).</p>
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<Accordion.Title className='text-white bg-accordion2 focus:bg-LGreen focus:ring-DMagenta focus:ring-2 hover:bg-LGreen hover:ring-white hover:ring-4 pb-2 text-xs'><p className='text-darkWhite'>Build <span className = 'text-right text-sm text-white underline'> Click to Expand</span></p></Accordion.Title>
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<p>After resuspending our <a className = 'italic'>Pantoea</a> microcin gBlocks, we created <a className='font-bold'>basic parts</a> of each microcin by cloning them into a high-copy GFP-dropout entry vector using GGA with the restriction enzyme BsmBI (Leonard et al., 2018). Once each basic microcin part was sequence confirmed, we used GGA again to assemble each basic microcin part with our CvaC15 part, the CP25 promoter, and the <a className='italic'>rpoC</a> terminator into a BTK low-copy backbone with the restriction enzyme BsaI (Leonard et al., 2018). The resulting assemblies formed constitutive microcin expression plasmids that followed our assembly scheme. Using whole-plasmid sequencing, we confirmed that the plasmid we designed was assembled correctly (Figure 7).</p><br></br>
<AccordianFigure imageSource='https://static.igem.wiki/teams/4579/wiki/phase-2-sequencing.png' figureLegend='Figure 7. Successful sequencing results for all 6 Pantoea microcin expression plasmids. The constructs that we designed in-silico is on top and the sequencing result is on the bottom. For reasons described in Figure 4, we manually examined mismatched regions and confirmed that they did not affect the transcriptional unit.'/><br></br>
<p>We then electroporated each <a className = 'italic'>Pantoea</a> microcin expression plasmid into a strain of <a className = 'italic'>E. coli</a> DH5α containing pSK01.</p><br></br>
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<p>To test whether the <a className = 'italic'>Pantoea</a> microcins were effective against any of the 4 pathogenic <a className = 'italic'>Pantoea</a> strains we had, we conducted ZOI assays against each pathogen using the <a className = 'italic'>E. coli</a> DH5α strains that we transformed in the build stage (Figure 8). For each of the 4 pathogens, we spotted a concentrated overnight culture of each strain (referred to as  “predator” strains) onto LB plates topped with a layer of soft agar containing the given pathogen.</p><br></br>
<AccordianFigure imageSource='https://static.igem.wiki/teams/4579/wiki/mcc04-mcc06-against-1600-1.png' figureLegend='Figure 8. Result of ZOI assays with E. coli DH5α (left) and P. vagans C9-1 (right, bottom) strains. Left: Mcc06 (1), microcin expression plasmid with pSK01 (2), neither plasmid (3). Right: Mcc04 (1), microcin expression plasmid with pSK01 (2), neither (3). Bottom:Mcc04 (1), microcin expression plasmid with pSK01 (2), neither (3) backlit for better visualize the presence of the ZOI. Created with BioRender.com.'/><br></br>
<p>After overnight incubation, it seemed that a small ZOI was produced by the Mcc04-secreting strain when it was spotted on <a className='italic'>P. agglomerans</a> PNG 92-11 and <a className='italic'>P. ananatis</a> PNA 97-1R. However, none of the other microcins produced apparent ZOIs against any of the pathogens.</p>
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<p>Since the ZOI generated by Mcc04 against <a className='italic'>P. agglomerans</a> PNG 92-11 and <a className='italic'>P. ananatis</a> PNA 97-1R. was very small, <a className='font-bold'>it was inconclusive whether the microcin was effective against those two strains</a>. After revisiting microcin testing literature and discussing our results with microcin researcher <a href = "https://2023.igem.wiki/austin-utexas/team" className='font-bold'>Jennifer Parker</a>, we learned that observing microcin effectiveness <a className='font-bold'>can be influenced by a number of factors</a> (Parker & Davies 2022). Over the course of the rest of the iGEM 2023 season, we went through multiple cycles of modifying our testing procedures to address the following factors:
</p><br></br>
<div className='grid grid-rows-auto border-gray-800 border-2 text-center bg-accordion1'>
              <div className= 'grid grid-cols-2 bg-DMagenta text-white p-2 text-center items-stretch'>
                <p className='px-6'>Microcin Effectiveness Factor</p>
                <p className='px-6'>Testing Modifications</p>
              </div>
              <div className= 'grid grid-cols-2 bg-white text-DMagenta p-2 text-center items-stretch'>
                <p className='px-6 overflow-wrap break-words'>Relative concentrations of the microcin secretor and target strains</p>
                <p className='px-6 overflow-wrap break-words'>Tried various combinations of prey and predator concentrations in ZOI </p>
              </div>
              <div className= 'grid grid-cols-2 bg-lavender text-DMagenta p-2 text-center items-stretch '>
              <p className='px-6 overflow-wrap break-words'>Access to the appropriate outer membrane receptor</p>
                <p className='px-6 overflow-wrap break-words'>Added dipyridyl (DIP), an iron chelator, to our ZOIs to see if increasing availability of iron uptake channels improved ZOI</p>
              </div>
              <div className= 'grid grid-cols-2 bg-white text-DMagenta p-2 text-center items-stretch '>
              <p className='px-6 overflow-wrap break-words'>Inhibition of the microcin secretor strain</p>
                <p className='px-6 overflow-wrap break-words'>Conducted growth curve assays to quantify self-inhibition of growth caused by Mcc04</p>
              </div>
              </div><br></br>
              <p>After first conducting growth curve assays with our <a className = "italic">E. coli</a> DH5α secretor strains, we chose to also transform our Mcc04 expression plasmid (along with pSK01) into one of our potential target strains: <a className = "italic">P. agglomerans</a> PNG 92-11. In doing so, we hoped to examine whether Mcc04 caused self-inhibition of growth in the target strain. When we conducted the growth curve with our <a className = "italic">P. agglomerans</a> PNG 92-11 strain expressing Mcc04, we observed slight growth inhibition (Figure 9).</p><br></br>
              <AccordianFigure
              imageSource='https://static.igem.wiki/teams/4579/wiki/mcc04-1597-growthcurve.png'
              figureLegend='Figure 9. Growth curve assay for self-inhibition with P. agglomerans PNG 92-11 strains expressing microcin plasmid backbone (yellow), Mcc02 (blue), or Mcc04 (red). All strains also express pSK01. The P. agglomerans PNG 92-11 strain expressing Mcc04 exhibited a visible degree of self-inhibition of growth.'/>
<p> These results led us to believe that Mcc04 may have weak antimicrobial activity against <a className='italic'>P. agglomerans</a> PNG 92-11. If this is true, self-inhibition of the <a className='italic'>P. agglomerans</a> strain can be lessened by <a className = 'font-bold'>providing an immunity protein</a> (Parker & Davies 2022). This led us to our most recent cycle which aimed to <a className = 'font-bold'>find and incorporate an immunity protein (IP) into our Mcc04 expression plasmid</a>.
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<p className='text-darkWhite'>
Most Recent Engineering Cycle<span className = 'flex justify-right text-sm text-white underline'> Click to Expand</span> </p>
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<p>To see if self-inhibition of the target strain caused by Mcc04 could be reduced by the presence of an immunity protein (IP), we decided to incorporate an IP into our Mcc04 expression plasmid. To find a likely IP for Mcc04, we manually searched the genome sequence of the strain of origin for open reading frames near Mcc04 and its native secretion system components (Parker & Davies 2022) (Figure 10). See the <a className='font-bold text-lavender underline hover:no-underline' href='https://2023.igem.wiki/austin-utexas/results'>Results Page</a> for more on how we found an IP candidate.</p><br></br>
<AccordianFigure
imageSource='https://static.igem.wiki/teams/4579/wiki/immunity-protein-in-the-genome.png'
figureLegend='Figure 10. Location of the immunity protein candidate for Mcc04 in the genome of a strain of origin. Created with BioRender.com.'/>
<p>To adapt the candidate to our assembly scheme, we designed a DNA sequence that consisted of Mcc04, its IP, and a ribosome binding site (RBS) (Figure 11) with BsmBI and BsaI restriction sites that produced the correct overhangs according to our GGA standard (Figure 3) (see the <a className='text-bold underline hover:no-underline text-lavender' href = 'https://2023.igem.wiki/austin-utexas/parts'>Parts Page</a> for more details). We then had the final sequence synthesized commercially as a Gene Fragment by Twist Biosciences.</p><br></br>
<AccordianFigure
imageSource='https://static.igem.wiki/teams/4579/wiki/mcc04-mcc04-ip.png'
figureLegend='Figure 11. Mcc04 expression plasmid versus Mcc04 + IP expression part. Created with BioRender.com.'/>
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<p>After resuspending our Mcc04+IP Gene Fragment, we used GGA with the restriction enzyme BsmBI to clone it into a high-copy GFP-dropout entry vector to create a basic part (Leonard et al., 2018). Once the basic part was sequence confirmed, we used GGA with the restriction enzyme BsaI to assemble the Mcc04+IP basic part with our CvaC15 part, the CP25 promoter, and the <a className='italic'>rpoC</a> terminator, into a BTK low-copy backbone (Leonard et al., 2018). This assembly resulted in a microcin expression plasmid that expressed both Mcc04 and its IP. Using whole plasmid sequencing, we confirmed that the plasmid we designed <a className='italic'>in silico</a> had been assembled correctly (Figure 12).</p><br></br>
<AccordianFigure
imageSource='https://static.igem.wiki/teams/4579/wiki/mcc04-ip-sequencing-results.png'
figureLegend='Figure 12. Successful sequencing results for Mcc04 with its immunity expression plasmid. The construct that we designed in silico is on top and the sequencing result is on the bottom.'/><br></br>
<p>We then electroporated the new plasmid into strains of <a className = "italic">E. coli</a> DH5α, <a className = "italic">E. coli</a> W3110, and <a className = "italic">P. agglomerans</a> PNG 92-11 containing pSK01.</p><br></br>
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<p>We conducted 14-hour growth curve assays to compare the growth of the <a className='italic'>P. agglomerans</a> PNG 92-11 Mcc04-secretor strains both with and without the IP (Figure 13). This allowed us to observe whether the growth inhibition of <a className='italic'>P. agglomerans</a> PNG 92-11 caused by Mcc04 on its own was remedied by providing immunity. This revealed whether the inhibition caused by Mcc04 (on its own) was due to antimicrobial activity of the microcin. </p> <br></br>
  <img src= 'https://static.igem.wiki/teams/4579/wiki/ipgrowthcurve.png' className='mx-15 w-full h-auto rounded-xl border-solid border-gray-800 border-2 drop-shadow-2xl'></img>
<p className='text-lavender italic text-center text-sm pt-2 pb-4'>Figure 13. Two replicate growth curves with P. agglomerans PNG 92-11 strains containing either our Mcc04 + IP expression plasmid (Gray), the Mcc04 expression plasmid without IP (Orange), or just the backbone of our modular microcin expression system (Blue). Each strain also contained pSK01. Averages of each time point are shown in bold while individual time points are grayed out. Time points were taken every half hour.</p><br></br>
<p></p><br></br>
<p>The results of repeated growth curve assays indicated that the growth of <a className='italic'>P. agglomerans</a> PNG 92-11 secreting Mcc04 was less inhibited when the IP was expressed.</p><br></br>

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<Accordion.Content className='bg-accordion4 text-darkWhite'>
<p>The growth curve results suggested that expression of the IP lessened inhibition of the Mcc04 secretor strain (Figure 13). This suggested that <a className='font-bold'>Mcc04 has promising antimicrobial activity against</a> <a className='font-bold italic'>P. agglomerans</a> <a className='font-bold'>PNG 92-11</a>. As such, we were able to learn that <a className='font-bold italic'>cinful</a> <a className='font-bold'>can be used to predict microcins that have antimicrobial activity against a pathogen of interest</a>. However, further testing and characterization are needed to fully confirm this.</p> <br></br>

<p>Additionally, the testing and assembly design changes we made allowed us to devise an <a className='font-bold'>improved process for finding and testing microcins</a>. We applied this improved process to find microcins against pathogenic strains of <a className = "italic">Erwinia amylovora</a> and <a className = "italic">Xanthomonas</a>. We cloned and assembled these microcins into microcin expression plasmids. However, we have yet to fully test them. See the <a className='font-bold text-lavender underline hover:no-underline' href='https://2023.igem.wiki/austin-utexas/results'>Results Page</a> for more information on these microcins. </p><br></br>

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<div id="Objective3"></div><h5></h5>

<h4></h4>
<section>Objective 3: Adapting Optimized Inducible Promoters for Our System</section>
<h3>We <a className='font-bold'>adapted a set of inducible promoter systems</a> that have been optimized in <a className='italic'>E. coli</a> (Meyer et al. 2019) for use in our microcin expression plasmid. This was accomplished by isolating the individual promoters and regulatory genes of the systems and applying our GGA standard to them, enabling them to form assemblies that follow our assembly scheme. We were able to demonstrate that the <a className = 'font-bold'>Ptet, PvanCC, and Pcin inducible promoters can properly promote GFP expression</a> when induced and limit it when uninduced, indicating that they could <a className = 'font-bold'>provide control over microcin expression within our microcin expression system</a>.</h3>
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Objective 3 Engineering Cycle  <span className = 'flex justify-right text-sm text-white underline'> Click to Expand</span> </p>
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<p>Because the original plasmids from Meyer et al. 2019 only differed in the promoter and regulatory gene being used, we designed two pairs of universal primers to amplify the promoter and regulatory gene of each system. The universal primers captured an RBS and a hammerhead ribozyme for each promoter and a terminator sequence upstream of each regulator. We ensured that the primers added BsmBI and BsaI restriction sites that produce overhangs in accordance with our GGA standard (Figure 14) (see the <a className='text-bold underline hover:no-underline text-lavender' href = 'https://2023.igem.wiki/austin-utexas/parts'>Parts Page</a> for more details). This way, the promoters would be compatible with our assembly scheme and therefore form versions of our microcin expression system under inducible control.</p><br></br>
<AccordianFigure
imageSource = 'https://static.igem.wiki/teams/4579/wiki/copy-of-part-gfp-1.png'
figureLegend = 'Figure 14. Inducible promoters and their regulatory gene parts defined according to our assembly scheme.'/>
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<p>Using the primers we designed, we performed PCR to amplify Ptet, Ptac, PluxB, PcymRC, pBAD, PvanCC, Pcin, and their corresponding regulatory genes: <a className='italic'>tetR</a>, <a className='italic'>lacI</a>, <a className='italic'>luxR</a>, <a className='italic'>cymR</a>, <a className='italic'>araC</a> and <a className='italic'>araE</a>, <a className='italic'>vanR</a>, and <a className='italic'>cinR</a>, respectively. Once we confirmed that the sizes of each of our parts were correct using gel electrophoresis, we purified them using the Zymo DNA Clean and Concentrate Kit.</p><br></br>
<p>We then created basic parts of each promoter and regulator by cloning each of them into a high-copy GFP-dropout entry vector (Leonard et al., 2018) using GGA with the restriction enzyme BsmBI. Once each basic part was sequence confirmed, we assembled Ptet, Ptac, PvanCC, and Pcin each with a BTK GFP coding sequence part and their corresponding regulator using GGA. We chose not to create assemblies with the other inducible promoters because we were unable to obtain the inducer molecules for them.</p><br></br>
<p> These assemblies resulted in GFP-expression plasmids that enabled us to examine the performance of the inducible promoters. Using whole plasmid sequencing, we confirmed that the plasmids we designed <a className='italic'>in silico</a> had all been assembled correctly (Figure 15).</p><br></br>
<AccordianFigure
imageSource='https://static.igem.wiki/teams/4579/wiki/inducible-proteins-sequencing.png'
figureLegend='Figure 15. Successful sequencing results for GFP assemblies with Ptac (A), PvanCC (B), Pcin (C), and Ptet (D) and their respective regulatory proteins. The construct that we designed in silico is on top and the sequencing result is on the bottom. Created with BioRender.com.'/>
<p>We then electroporated each plasmid into the strains of <a className='italic'>P. agglomerans</a> PNG 92-11 and <a className='italic'>E. coli</a> DH5α containing pSK01 we created while pursuing <a className='text-lavender font-bold underline hover:no-underline' href = "#Objective2">Objective 2</a>. This resulted in a strain that utilized the new inducible promoters while maintaining similar expression to the full two-plasmid system we are using.</p><br></br>
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<p>We performed a fluorescence assay on our strains containing the Ptet, PvanCC, and Pcin-controlled GFP-expression plasmids and pSK01 (Figure 16). We grew induced and uninduced cultures of the strains overnight and took a single endpoint fluorescence reading for each culture. When taking our readings, we set excitation and emission to 485 nm and 535 nm, respectively.</p><br></br>
<img src= 'https://static.igem.wiki/teams/4579/wiki/fluorescence-assays.png' className='mx-15 w-full h-auto rounded-xl border-solid border-gray-800 border-2 drop-shadow-2xl'></img>
<p className='text-lavender italic text-center text-sm pt-2 pb-4'>Figure 16. GFP fluorescence in E. coli DH5α and P. agglomerans PNG 92-11 under the inducible promoters Ptet, Ptac, PvanCC, and Pcin. Ptac was not tested in P. agglomerans PNG 92-11 due to time. Induced (+) cultures exhibited much greater fluorescence than uninduced ones, indicating that the promoters have a clear “on/off” state. Created with BioRender.com.</p>
<br></br>
<p>Our endpoint readings indicated that the induced and uninduced states of each promoter caused significantly different levels of GFP expression.</p><br></br>
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<p>Through our assay results, we demonstrated that the inducible systems with Ptet, Ptac, PvanCC, Pcin, and their corresponding regulators were <a className='font-bold'>still functional when adapted to our assembly scheme and system</a>. We found that the inducible systems were also functional in <a className='italic'>P. agglomerans</a> PNG 92-11. This indicated that the system is likely also functional in <a className='italic'>Pantoea</a> strains. From this, we learned that these <a className='font-bold'>inducible promoters should enable us to control microcin expression</a> in our system. As such, <a className='font-bold'>we chose to make new assemblies with MccV and Mcc04 under inducible expression</a> using these promoters. While we successfully assembled these inducible microcin constructs, we were unable to test them in ZOI or growth curve assays due to lack of time.</p><br></br>
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<h5></h5>

<h4></h4>
<section>References</section>
<Sources
citationText='Cole, T. J., Parker, J. K., Feller, A. L., Wilke, C. O., & Davies, B. W. (2022). Evidence for widespread class II microcins in Enterobacterales Genomes. Applied and Environmental Microbiology, 88(23), e01486-22.'
doi=''/>
<Sources
citationText='Engler, C., Romy Kandzia, & Sylvestre Marillonnet. (2008). A One Pot, One Step, Precision Cloning Method with High Throughput Capability. PLOS ONE, 3(11), e3647–e3647.'
doi='https://doi.org/10.1371/journal.pone.0003647 '/>
<Sources
citationText='Engler, C., Gruetzner, R., Romy Kandzia, & Sylvestre Marillonnet. (2009). Golden Gate Shuffling: A One-Pot DNA Shuffling Method Based on Type IIs Restriction Enzymes. PLOS ONE, 4(5), e5553–e5553. '
doi='https://doi.org/10.1371/journal.pone.0005553 '/>
<Sources
citationText='Kim, S.-Y., Parker, J., Gonzalez-Magaldi, M., Telford, M. S., Leahy, D. J., & Bryan William Davies. (2023). Export of diverse and bioactive peptides through a type I secretion system. BioRxiv (Cold Spring Harbor Laboratory).'
doi='https://doi.org/10.1101/2023.01.26.525739'/>
<Sources
citationText='Madeira, F., Pearce, M., Tivey, A., Prasad Basutkar, Lee, J., Ossama Edbali, Nandana Madhusoodanan, Kolesnikov, A., & López, R. (2022). Search and sequence analysis tools services from EMBL-EBI in 2022. Nucleic Acids Research, 50(W1), W276–W279.'
doi='https://doi.org/10.1093/nar/gkac240 '/>
<Sources
citationText='Meyer, A.J., Segall-Shapiro, T. H., Glassey, E., Zhang, J., & Voigt, C. A. (2018). Escherichia coli “Marionette” strains with 12 highly optimized small-molecule sensors. Nature Chemical Biology, 15(2), 196–204.'
doi='https://doi.org/10.1038/s41589-018-0168-3'/>
<Sources
citationText='Parker, J. D., & Bryan William Davies. (2022). Microcins reveal natural mechanisms of bacterial manipulation to inform therapeutic development. Microbiology, 168(4).'
doi='https://doi.org/10.1099/mic.0.001175'/>
<h5></h5>
<Footer Year='2023'/>
</>
)
}

export default engineering


import Figures from '../components/Figures';
import Footer from '../components/Footer';
import Navigation from '../components/Navigation';
import Sources from '../components/Sources';
import Head from 'next/head';

const results = () => {
return (

<>

<Navigation name='UT Austin iGEM 2023'/>

<Head>
    <title>Preliminary Results</title>
    <link rel="shortcut icon" href="https://static.igem.wiki/teams/4579/wiki/logo-removebg-preview.png" />
</Head>

<h1 className="animate-fade-up">Results</h1>

<h4></h4>
<section>Introduction</section>
<h3>To achieve our objective of engineering a modular microcin expression system to target and kill bacteria plant pathogens, we established the following project pipeline:</h3>

<h3>
<ul className="list-decimal ml-6">
    <li><a className='text-DMagenta underline hover:no-underline' href="#Testing">Design and test a modular microcin expression system plasmid that allows for promoters,  microcins, and immunity proteins to be swapped in or out of the construct</a> </li>
    <li> <a className='text-DMagenta underline hover:no-underline' href="#FindNovel">Find and characterize novel microcins and their associated immunity proteins using computational methods</a></li>
    <li><a className='text-DMagenta underline hover:no-underline' href="#Exhibiting">Perform bioassays to demonstrate pathogenesis of pathogenic strains used to test microcins and develop an <span className="italic">in vivo</span> assay for testing the modular microcin expression system</a></li>
</ul>
</h3>
<h3>As we progressed through this pipeline we obtained significant results that showed the identification of novel microcins, function and effectiveness of our microcin expression system, and pathogenicity of our target pathogens.</h3>
<div id="Testing"></div><h5></h5>

<h4></h4>
<section>Testing Our Modular Microcin Expression System </section>
<h3>In order to develop our system, we first needed to take the original expression system from Kim et. al and adapt it to be modular (Figure 1). To confirm this, we cloned Microcin V (MccV), a known and well characterized microcin, into our microcin expressing plasmid and tested for its presence (Figure 2). MccV is natively secreted by <a className="italic">E. coli</a> that targets and kills other susceptible <a className="italic">E. coli</a> lacking its immunity protein (Parker & Davies, 2022).</h3>

<h3>
<p className=" flex justify-center pt-4 pb-2"><img src="https://static.igem.wiki/teams/4579/wiki/our-system-vs-davies.png" className="mx-15 w-2/3 h-auto rounded-xl border-solid border-gray-800 border-2 drop-shadow-2xl"/></p>
<p className="text-Avocado italic text-center pt-2 pb-4">Figure 1. The original MccV expression plasmid versus the general assembly schema for our modular microcin expression plasmid. Created with BioRender.com.</p>
</h3>

<h3>As a positive control, we used a strain called <a className="italic">E. coli</a> W3110 SK01 that contains microcin plasmid pSKP00 with genes for MccV and a secretion system plasmid pSK01 from Kim et al., 2022. <a className='font-bold'>This strain has been shown to successfully secrete MccV against susceptible strains of </a><a className="italic font-bold">E. coli</a>. We made a plate with <a className="italic">E. coli</a> W3110 as the ‘prey’ lawn and compared our modular microcin expressing assembly against the positive control.</h3>
<Figures
    imageSource="https://static.igem.wiki/teams/4579/wiki/results-figure-2.jpg"
    figureLegend="Figure 2: Testing our modular microcin expressing plasmid by making it ssecrete a well-characterized microcin to be compared against a positive control secreting the same microcin. 1) Our modular microcin expressing plasmid coding for MccV and a secretion system plasmid. 2) The positive control strain expressing MccV and secretion system. 3) Our modular microcin expressing plasmid coding for MccV without the secretion system. 4) The positive control with MccV without the secretion system. 5) The empty chassis of our construct. 6) The empty chassis for the positive control "
/>
<h3>Upon observing a clearing for our microcin secreting construct in comparison to the positive control, we proved that <a className="font-bold">our microcin expressing assembly plasmid is effective at secreting microcins when coupled with the secretion system plasmid.</a></h3>

<section>Characterizing Potential Inducers</section>
<h3>Our tests so far have been using constitutive promoters to regulate microcin secretion which might make our experiments miss some results. This is because the chassis has no way to stop production of the toxic protein and thus might be dying before secreting the amount of microcin that can be observable. To expand upon the control of the expression of our microcins, we have opted to develop several regulated plasmids in addition to the constitutive expression plasmids we have been using for testing. We characterized several promoters and regulatory genes sourced from the <a className="italic">E. coli</a> 'Marionette" study (Meyer et al., 2019). Firstly, we tested expression of green fluorescent protein (GFP) using these promoters transformed into <a className="italic">P. ananatis</a> PNA 97-1R and <a className="italic">E. coli</a> DH5α and compared their fluorescence in induced and uninduced cultures. By showing promoter expression in two different chassis, we can show that these promoters are functional across different chassis and thus, can be used as a component in the modular microcin expression plasmid when it is transformed in a biocontrol or <a className="italic">E. coli.</a> </h3>
<h3 className = 'flex flex-row justify-center pt-4 pb-2'>
<img src="https://static.igem.wiki/teams/4579/wiki/figure-10-left.jpg" className="rounded-l-xl w-[45%]"/>
<img src="https://static.igem.wiki/teams/4579/wiki/figure-10-right.jpg" className="rounded-r-xl w-[45%]"/>
</h3>
<h3 className = 'text-Avocado italic text-center pt-2 pb-4'>Figure 3: Graphs characterizing expression of various promoters based on florescence of GFP for pTet (anhydrotetracycline regulated via TetR), pTac (IPTG regulated via LacI), pVanCC (Vanillic acid regulated via VanR) and pCin (OHC-14 regulated via CinR). Left: Promoters transformed into P. ananatis PNA 97-1R. Right: Promoters transformed into E. coli DH5α.</h3>
<h3>Based on the clear high expression upon induction of the promoters (Figure 3), <a className="font-bold">we were able to successfully characterize the following promoters based on their GFP fluorescence:</a></h3>
<h3>
<ul className="list-decimal ml-[8%]">
    <li>pTet (anhydrotetracycline regulated via TetR)</li>
    <li>pTac (IPTG regulated via LacI) </li>
    <li>pVanCC (Vanillic acid regulated via VanR)</li>
    <li>pCin (OHC-14 regulated via CinR)</li>
</ul>
</h3>
<div id="FindNovel"></div><h5></h5>

<h4></h4>
<section>Finding Novel Microcins</section>
<h3>To find and characterize novel, putative microcins, we developed a multi-stage pipeline that ranged from computational analysis to laboratory benchwork (Figure 4).</h3>
<Figures
    imageSource='https://static.igem.wiki/teams/4579/wiki/screenshot-2023-10-12-092057.png'
    figureLegend='Figure 4: Our pipeline depicting our project to search and test putative, novel microcins from computational output to biological assays.'
/>
<h3>We began our project with finding putative microcins that could potentially target onion pathogens from the <a className="italic">Pantoea</a> genus using a microcin identification pipeline called <a className="italic">cinful</a>. After passing <a className="italic">Pantoea</a> genomes through <a className="italic">cinful</a>, the software provided an output of potential microcin sequences found within those genomes based on a model the software constructs from existing, characterized microcins (Cole et al., 2022). Out of the 1337 <a className="italic">Pantoea</a> we screened, <a className="font-bold">we identified 6 unique microcins</a> along with the species they originate from:</h3>
<h3>
<ul className="list-decimal ml-6">
    <li><a className="font-bold">Mcc01</a> – <a className="italic">Pantoea sp.</a> PSINH3 </li>
    <li><a className="font-bold">Mcc02</a> – <a className="italic">Pantoea ananatis</a> </li>
    <li><a className="font-bold">Mcc03</a> – <a className="italic">Pantoea vagans</a></li>
    <li><a className="font-bold">Mcc04</a> – <a className="italic">Pantoea vagans</a> </li>
    <li><a className="font-bold">Mcc05</a> - <a className="italic">Pantoea sp.</a> PSINH3</li>
    <li><a className="font-bold">Mcc06</a> – <a className="italic">Pantoea vagans</a></li>
</ul>
</h3>
<h3><a className="italic">cinful</a> also outputs the start and stop position of the microcin sequence in the genome. Using this knowledge, <a className="font-bold">we identified immunity proteins for each of the microcins</a> by looking at open-reading frames using a genome sequence viewer (such as SnapGene) around the microcin based on the following criteria:
</h3>
<h3>
<ul className="list-decimal ml-[8%]">
    <li>Protein is found on the same sense, either upstream or downstream of the microcin sequence</li>
    <li>Has a nucleotide length between 300-600 base pairs</li>
    <li>The annotation of the open reading frame is ‘hypothetical protein’ and not that of a secretion system component (such as <a className="italic">TolC</a> or Membrane Fusion Protein)</li>
</ul>
</h3>
<h3>For our reference, we constructed a sequence alignment tree (Figure 5) of our chosen microcins to classify the similarities between amino acid sequences. <a className="font-bold">We predicted that the microcin with highest sequence divergence from the rest of the identified microcins would exhibit the most toxicity towards the tested genus.</a> This occurs because native immunity proteins typically only confer resistance to their associated microcin and do not protect against microcins with highly differing sequences.</h3>
<Figures
    imageSource="https://static.igem.wiki/teams/4579/wiki/results-figure-1.png"
    figureLegend="Figure 5: Nucleotide sequence alignment tree showing degree of similarity between chosen microcins. Each cluster contains the genome that the microcin originates from. This diagram helps determine which microcins chosen from the Pantoea genus are the most divergent to identify the most effective microcin "
/>
<h3>The sequence alignment chart produced by <a className="italic">cinful</a> expresses how similar potential microcin sequences are to each other through distance, with the most similar sequences being closest. The sequence alignment chart for the screened <a className="italic">Pantoea</a> species suggested that <a className="font-bold">Mcc02 had the most divergent sequence</a> from the other possible microcins. </h3>
<h3>To show versatility and modularity of our secretion system, we wanted to test multiple microcins from different origins. Thus, <a className="font-bold">we also screened microcins whose origins are from </a><a className="font-bold italic">Erwinia</a> <a className="font-bold">and</a> <a className="font-bold italic">Xanthomonas</a><a className="font-bold"> genera</a> (Table 1). Due to the lack of time, we could only find immunity proteins for some of these microcins.</h3>

<h3>
<p className=" flex justify-center pt-4 pb-2"><img src="https://static.igem.wiki/teams/4579/wiki/new-microcin-2.png" className="mx-2 border-solid border-gray-800 border-2 drop-shadow-2xl"/></p>
<p className="text-Avocado italic text-center pt-2 pb-4">Table 1: This table represents amino acid sequences for putative microcins from Erwinia and Xanthomonas genera</p>
</h3>

<h3>We plan to test these putative microcins in the future to further validate our modular microcin expression system and our microcin and immunity protein screening pipeline.</h3>

<section>Showing Putative Microcin Effectiveness</section>
<h3>To demonstrate if our putative, novel microcins exhibit antibacterial properties against their predicted target, we first conducted a zone of inhibition assay. Since our microcins were selected such that they target bacteria from the <a className="italic">Pantoea</a> genus, <a className="font-bold">we conducted zone of inhibition assays with our microcins against pathogenic </a><a className="italic font-bold">Pantoea</a> <a className="font-bold">strains</a>. The pathogenic <a className="italic">Pantoea</a> tested were: </h3>
<h3>
<ul className="list-decimal ml-6">
    <li><a className="italic">Pantoea agglomerans</a> PNG 92-11</li>
    <li><a className="italic">Pantoea allii</a> PNA 200-100</li>
    <li><a className="italic">Pantoea ananatis</a> LMG 2665</li>
    <li><a className="italic">Pantoea ananatis</a> PNA 97-1R</li>
</ul>
</h3>
<h3>Since we want our microcins to be able to target and kill pathogenic strains that cause onion center rot, we conducted onion rot assays (check <a className="font-bold underline hover:no-underline text-DMagenta" href="#Exhibiting">Exhibiting Onion Bacteria Pathogenicity</a>) to prove that the pathogenic strains we use for testing impart the symptoms associated with onion center rot.</h3>
<h3 className = 'flex flex-col justify-center pt-4 pb-2'>
<img src="https://static.igem.wiki/teams/4579/wiki/results-figure-3-top.jpg" className="mx-[15%] rounded-t-xl border-solid border-2 border-BGreen"/>
<img src="https://static.igem.wiki/teams/4579/wiki/results-figure-3-middle.jpg" className="mx-[15%]"/>
<img src="https://static.igem.wiki/teams/4579/wiki/results-figure-3-last.jpg" className="mx-[15%] rounded-b-xl border-solid border-2 border-BGreen"/>
</h3>
<h3 className = 'text-Avocado italic text-center pt-2 pb-4'> Figure 6: Zone of inhibition assay to test putative, novel microcin against their predicted targets, Pantoea. Top: Positive result, ZOI plate with P. ananatis PNA 97-1R as prey and transformed E.coli DH5α as predator. Modular microcin-expressing plasmid Mcc04 with secretion system plasmid (1), modular microcin-expressing plasmid  Mcc04 without secretion system plasmid (2) untransformed strain (3). Middle: Two spots showing effect of secretion system on Mcc04 against P. agglomerans PNG 92-11. Bottom: Negative result, Zone of inhibition plate with with P. ananatis PNA 97-1R as prey and transformed E.coli DH5α as predator. Modular microcin-expressing plasmid Mcc06 with secretion system plasmid (1), modular microcin-expressing plasmid Mcc06 without secretion system plasmid (2), untransformed strain (3).</h3>

<h3>Out of the 6 microcins we selected from the <a className="italic">Pantoea</a> genome, we noticed that Mcc04 against <a className="italic">Pantoea ananatis</a> PNA 97-1R showed the best measure of clearing out of all the microcin-pathogen combinations during the first round of experimenting. <a className="font-bold"> This clearing indicates around the microcin secreting strain that our putative microcin is effective in killing</a><a className="italic font-bold"> P. ananatis</a><a className="font-bold"> PNA 97-1R</a>. Mcc04 against a lawn of <a className="italic"> Pantoea agglomerans</a> PNG 92-11 also showed a slight clearing around the microcin secreting strain as well (Figure 6). The clearing around these microcin secreting strains is not as dramatic as the ones shown for MccV. However, it should be restated that the ideal conditions under which these putative microcins are secreted is subject to further testing. We could not replicate the above zones we saw for Mcc04, nor did we have any positive results with other microcins. Thus, we decided to test microcin expression using a more sensitive, quantitative assay: performing growth curves. </h3>
<h3>We conducted growth curve assays with our modular microcin expression system transformed into pathogenic <a className="italic">Pantoea</a> strains such that if we observed an inhibition in growth, we could conclude that the microcin targets that <a className="italic">Pantoea strain</a>. To explain how we could obtain a culture of a strain that secretes a protein that can kill itself, we transformed the secretion system plasmid first into the chassis and then transformed the microcin expressing plasmid. To keep the metabolic burden of two plasmids and having a secretion system consistent, our controls contain the secretion system plasmid and the backbone of our modular microcin expressing plasmid. Out of all our putative microcins, <a className='font-bold'>we chose Mcc04 and Mcc02 as our candidates for doing a growth curve</a> given Mcc04’s previous success and confidence in Mcc02’s neighboring genomic elements that help determining if the sequence is a microcin or not and its extent of difference in sequence as seen from the sequence alignment tree.</h3>

<h3 className = 'flex flex-col justify-center pt-4 pb-2'>
<img src="https://static.igem.wiki/teams/4579/wiki/results-figure-5.jpg" className="rounded-xl border-solid border-2 border-BGreen"/></h3>
<h3 className = 'text-Avocado italic text-center pt-2 pb-4'>Figure 7: Growth curves for pathogenic Pantoea strains transformed with our modular microcin expression system to observe self-inhibition of growth. The pathogenic Pantoea strains transformed with the modular microcin expressing strain were A) P. agglomerans PNG 92-11 B) P. allii PNA 200-100 C) P. ananatis LMG 2665"</h3>

<h3>As seen from the graph for <a className="italic"> P. ananatis </a>LMG 2665, we observed that the growth curve for the microcin expressing strain has markedly downward shift compared to the strain not expressing any microcins (Figure 7). This indicates an inhibition in growth caused by the microcin, providing evidence that <a className="font-bold">our putative, novel microcins exhibit antibacterial properties against their predicted targets while also proving that our microcin screening method is effective.</a></h3>
<section>Showing Putative Microcin and Immunity Protein Effectiveness </section>
<h3>Finding the right immunity protein for a microcin is crucial for a chosen chassis to effectively secrete microcins, even if the microcin does not target the chassis since a microcin is a toxic protein regardless (Parker & Davies, 2022). Since Mcc04 has been giving us the best result in targeting and killing pathogenic <a className="italic">Pantoea</a> strains, we cloned in the immunity protein we predicted for Mcc04. We conducted a zone of inhibition assay for <a className="italic">E.coli</a> containing Mcc04 and its immunity protein against <a className="italic">Pantoea ananatis</a> PNA 97-1R, which had previously shown a zone without the immunity protein. <a className="font-bold">We expected the zone formed with the microcin coupled with the immunity protein to be larger and more distinct than the one we found for the strain with only the microcin</a>. This is because the secretion of the microcin should be improved by the presence of an immunity protein.</h3>

<Figures
    imageSource='https://static.igem.wiki/teams/4579/wiki/results-figure-6.jpg'
    figureLegend='Figure 8: Zone of Inhibition plate to test our Pantoea microcin and immunity protein assembly against Pantoea ananatis PNA 97-1R. A) E. coli DH5α with the modular Mcc04 and immunity protein expressing plasmid along with the secretion system plasmid B) E. coli DH5α with only microcin backbone and secretion system. C) E. coli DH5α by itself (without microcin, immunity protein or secretion system)'
/>
<h3>As seen from the plate, there was no distinct zone of inhibition around the microcin and immunity protein expressing strain (Figure 8). This could either be an indication that the immunity protein is not effective or the microcin is not being secreted and diffused properly into the medium. Thus, we decided once again to test the microcin and immunity protein with the <a className="font-bold">growth curve assay</a> as it provides a sensitive, quantitative measurement that would also minimize the problem of the microcin not diffusing properly.</h3>
<h3><a className="font-bold">We expected the graph of the strain expressing the immunity protein to show higher growth than the strain expressing only the microcin</a> because the immunity protein confers resistance towards the microcin, making the chassis less susceptible to the microcin’s toxic effects. We transformed <a className="italic">P. agglomerans</a> PNG 92-11 with both our modular Mcc04 and immunity protein expressing plasmid and the secretion system plasmid then conducted a 14 hour growth curve along with microcin only expressing plasmid as the positive control (Figure 9). </h3>

<h3 className = 'flex flex-col justify-center pt-4 pb-2'>
<img src="https://static.igem.wiki/teams/4579/wiki/results-figure-7.jpg" className="rounded-xl border-solid border-2 border-BGreen"/></h3>
<h3 className = 'text-Avocado italic text-center pt-2 pb-4'>Figure 9: Two repeats of the growth curve assay done on two different days to show activity of P. agglomerans PNG 92-11 containing the modular microcin and immunity protein expressing assembly. Each faded curve is a replicate of an individual colony of the strain; there are 8 total replicates for each strain. The orange growth curve is the strain containing the microcin and immunity protein assembly and secretion system; blue growth curve is the strain containing only the microcin assembly and secretion system; grey growth curve is the strain containing microcin back bone and secretion system (negative control)</h3>

<h3>For the first repeat of the growth curve, the microcin only expressing curve shows a downward shift starting from the log phase from the negative control curve proving that the microcin shows effective self-inhibition in the absence of an immunity protein. The microcin and immunity protein expressing curve is shown significantly higher than that for the microcin only expressing curve <a className="font-bold">proving that our immunity protein is effective at imparting resistance against the microcin for the chassis</a>. For the second repeat of the experiment, we got the same results except for the microcin only expressing curve starting its downward shift from the negative control much later. This discrepancy might be explained by the fact that these curves were conducted on two different days with different sets of colonies. With more repeats of this experiment, we can be sure what behavior to expect of Mcc04.</h3>
<h3>Overall, since the microcin only expressing graph shows a clear downward shift indicating slower growth compared to the control and the strain with the immunity protein, <a className="font-bold">our predicted immunity protein is effective with our putative microcin which also provides proof that our immunity protein screening method is also effective.</a></h3>
<h3>All assemblies of our modular microcin expressing system that were made for testing are sequence verified. For details visit the <a className="font-bold underline text-DMagenta hover:no-underline" href="https://2023.igem.wiki/austin-utexas/engineering">engineering page</a></h3>
<div id="Exhibiting"></div><h5></h5>

<h4></h4>
<section>Exhibiting Onion Bacteria Pathogenicity</section>
<h3>Although the <a className="italic">Pantoea</a> strains we used belonged to species typically associated with onion center rot, we still recognized the need to characterize their virulence levels. Upon reviewing the relevant literature, we found that two of our strains were found to have gene clusters associated with the virulence of the rot (Stice et al., 2021). <a className="font-bold">To characterize the pathogenicity of our pathogenic</a> <a className="italic">Pantoea</a><a className="font-bold"> strains that we would be testing our microcins with, we conducted onion rot assays</a>. These assays also allowed us to develop a potential in vivo assay to test the effectiveness of our modular microcin expression system against onion pathogens. For the onion rot assay, we injected separate Texas 1015Y Sweet onions with one of our four pathogenic <a className="italic">Pantoea</a> strains along with a water control. We incubated all onions at 37°C for 14 days, then observed the rot formed by cutting open each onion at the center. The severity of browning and softening of onion center flesh compared to the water control was observed to evaluate the virulence of each strain.</h3>
<Figures
    imageSource='https://static.igem.wiki/teams/4579/wiki/figure-7.jpeg'
    figureLegend='Figure 10: Photos of the onions cut after 14 days that were inoculated at different optical densities (OD) along with a water control '
/>
<h3>We observed that <a className="italic">P. ananatis</a> PNA 97-1R showed the most virulent pathogenic properties compared to all other strains given that it showed the most browning and softening of flesh after 14 days of inoculation. Based on our microcin testing, <a className="font-bold">one of our putative microcins (Mcc04) is effective at killing the same strain that causes the most pathogenicity out of all the pathogens we are testing.</a> Concluding <a className="italic">P. ananatis</a> PNA 97-1R as our most virulent strain, we then planned to characterize the timeline of the rot for this strain as a reference to use when we conduct in-vivo tests of our modular microcin expressing system. We conducted an onion rot progression assay in which multiple onions were inoculated on the same day with <a className="italic">P. ananatis</a> PNA 97-1R and were then sequentially cut over 28 days. This allowed us to determine what timeline would be used for our planned in vivo assays.</h3>
<Figures
    imageSource='https://static.igem.wiki/teams/4579/wiki/figure-8.jpg'
    figureLegend='Figure 11: Photos of different onions inoculated on the same day with P. ananatis PNA 97-1R, E. coli W3110 and water cut on different days.'
/>
<h3>Using our onion assays, we were able to successfully confirm and characterize the pathogenicity of our <a className="italic">Pantoea</a> strains as well as develop a potential framework for testing the effectiveness of our microcin expression system against onion pathogens in vivo.</h3>
<div id="Characterizing"></div><h5></h5>

<h4></h4>
<section>Conclusion</section>
<h3>Based on our goals highlighted earlier, we list the following as what we have achieved for our research project:</h3>
<h3>
<ul className="list-decimal ml-[8%]">
    <li className="whitespace-break-spaces"><a className="underline">Design and test a modular microcin expression system plasmid that allows for promoters, microcins and immunity proteins to be swapped in or out</a> – <br></br>Our assembly of a modular microcin expression system was able to secrete various microcins (well-known and our putative microcins) when paired with a secretion system plasmid. We also characterized inducible promoters that can be swapped into the microcin expression system for future testing</li>
    <li><a className="underline">Find and characterize novel microcins and associated immunity proteins using computational and microbiological methods</a> –<br></br>
    We found microcins and associated immunity proteins present in the <a className="italic">Pantoea</a> genus such that when expressed can target and kill pathogenic <a className="italic">Pantoea</a> strains that cause onion center rot. We also characterized these microcins based on their sequence alignment tree to identify the microcin with the most different sequence. Microcins and immunity proteins from other genera (<a className="italic">Erwinia</a> and <a className="italic">Xanthomonas</a>) were also found. We found one of our putative microcins effective against two of our pathogenic <a className="italic">Pantoea</a> strains along with an associated immunity protein that imparts resistance to the chassis
    </li>
    <li><a className="underline">Perform a bioassay to demonstrate pathogenesis of pathogenic strains used to test microcins and develop an in-vivo assay for testing the modular microcin expression system</a> –<br></br>
    We conducted a multi-day onion rot assay that helped characterize the pathogenicity of the pathogenic strains of <a className="italic">Pantoea</a> we are using for testing. The strain that we found the most pathogenic was susceptible to one of our putative microcins.
    </li>
</ul>
</h3>
<h3>Here are our future goals and testing plans for our project:</h3>
<h3>
<ul className="list-decimal ml-[8%]">
    <li>Make our two-plasmid secretion system in broad-host range backbones such that it can be inserted into existing biocontrols </li>
    <li>Test microcins from other genera with our system to further validate the modularity of our microcin expressing system</li>
    <li>Test our microcins using characterized inducible promoters which would allow customization</li>
    <li>Utilize our characterization of <a className="italic">P. ananatis</a> PNA 97-1R virulence and rot progression to visualize the potency of our modular microcin expression system in-vivo</li>
</ul>
</h3>
<h5></h5>

<h4></h4>
<section> References </section>
<Sources
citationText='Cole, T. J., Parker, J. K., Feller, A. L., Wilke, C. O., & Davies, B. W. Evidence for Widespread Class II Microcins in Enterobacterales Genomes. Applied and Environmental Microbiology, 2022, 88(23).'
doi = 'https://doi.org/10.1128/aem.01486-22'
/>
<Sources
citationText='Kim, S-Y., Parker, J. K., Gonzalez-Magaldi, M., Telford, M. S., Leahy, D. J., & Davies, B. W. (2023). Export of Diverse and Bioactive Small Proteins through a Type I Secretion System. Applied and Environmental Microbiology, 89(5), e00335-23.'
doi = 'https://doi.org/10.1128/aem.00335-23'
/>
<Sources
citationText='Meyer, A. J., Segall-Shapiro, T. H., Glassey, E., Zhang, J., & Voigt, C. A. (2019). Escherichia coli “Marionette” strains with 12 highly optimized small-molecule sensors. Nature Chemical Biology, 15(2), 196-204.'
doi = 'https://doi.org/10.1038/s41589-018-0168-3'
/>
<Sources
citationText='Parker, J. K., & Davies, B. W. (2022). Microcins reveal natural mechanisms of bacterial manipulation to inform therapeutic development. Microbiology, 168(4), 001175.'
doi = 'https://doi.org/10.1099/mic.0.001175'
/>
<Sources
citationText='Stice, S. P., Shin, G. Y., de Armas, S., Koirala, S., Galván, G. A., Siri, M. I., Severns, P. M., Coutinho, T., Dutta, B., & Kvitko, B. H. (2021). The Distribution of Onion Virulence Gene Clusters Among Pantoea spp. Frontiers in Plant Science, 12.'
doi = 'https://doi.org/10.3389/fpls.2021.643787'
/>
<h5></h5>
<Footer Year='2023' />
</>
)
}

export default results