Difference between revisions of "Team:Fudan/Results"

(after vgj)
(Undo revision 379142 by Wave (talk))
 
Line 210: Line 210:
 
     <div class="col m2 l3 hide-on-small-only">&nbsp;</div>
 
     <div class="col m2 l3 hide-on-small-only">&nbsp;</div>
 
     <div class="col s12 m8 l6">
 
     <div class="col s12 m8 l6">
−
       <img src="https://static.igem.org/mediawiki/2020/e/eb/T--Fudan--title_safety.svg" alt="safety" style="width:100%">
+
       <img src="https://static.igem.org/mediawiki/2020/5/50/T--Fudan--title_results.svg" alt="results" style="width:100%">
 
       </div>
 
       </div>
 
   </div>
 
   </div>
Line 221: Line 221:
 
               html代码不在乎多个空格，仅显示一个
 
               html代码不在乎多个空格，仅显示一个
 
         -->
 
         -->
−
  <h1 tabIndex="1">The most primary issue to consider</h1>
+
<h2>Improvement of the antimicrobial peptide expressing system</h2>
 
   <div class="column full_size">
 
   <div class="column full_size">
−
     <p>Safety is always the most primary issue to consider, especially for projects in which potential consumers are the vulnerable elderly. The risks for everyone who has access to engineered probiotic from production to consumption should be carefully identified and managed.</p>
+
     <p>Here, we tried to improve the former antimicrobial peptide (Mccb17) expression, based on 2019 Fudan <a href="http://parts.igem.org/Part:BBa_K3245010">BBa_K3245010</a>. By dividing into the peptide expressing part and the immunity part, we firstly tested whether the polycistron could work properly and separately, then tried to manipulate each of their expression levels for better results.
 +
</p>
 
   </div>
 
   </div>
  
−
  <div class="clear"></div><!-- 一个章节结束需要clear来留白 如果觉得内容间差异大再加上extra_space -->
+
<div class="clear"></div><!-- 一个章节结束需要clear来留白 如果觉得内容间差异大再加上extra_space -->
  
−
   <h2>Laboratory Safety</h2>
+
   <div class="column full_size">
−
    <div class="column full_size" >
+
  <h4>Verification of the expression</h4>
−
     <p>The Fudan team is fully compliant with the safety and security policies of the iGEM. Safety has been carefully and thoroughly considered throughout our project design and experimental operation.</p></div>
+
     <p>First, to affirm the system could work properly, we started from the very beginning by testing the expression of each mcb gene in the cluster as well as of the polycistrons in each part. We constructed 10 plasmids (with pGEX backbone adapted to perform IPTG induced expression), containing <i>mcbA/ mcbB/ mcbC/ mcbD/ mcbE/ mcbF/ mcbG/ mcbABCD/ mcbBCD/ mcbBC</i> respectively, transformed successfully them into <i>E. coli</i> BL21. We have made further confirmation that every part can be induced successfully due to the correct bands on the SDS-PAGE gels, only shown in IPTG induced samples. If needed, we could purify these protein products via immobilized Ni affinity chromatography (GST was replaced with His-tag in our constructs).
−
  <div class="clear"></div><!-- 一个章节结束需要clear来留白 如果觉得内容间差异大再加上extra_space -->
+
    </p>
 +
  </div>
  
−
  <div class="column full_size" >
+
<div class="column full_size">
 +
<h4>Verification of the function</h4>
 +
<p>As <a href="/Team:Fudan/Design">our design page</a> describes, McbABCD should show a certain degree of restrain to both the growth and metabolism of its co-cultured cells. While with McbEFG, engineered <i>E. coli</i> are supposed to have better immunity themselves against the MccB17 coded by <i>mcbABCD</i>, so as to gain advantage, affect the surrounding WT’s metabolism (expressing of GFP in our experiments), and better survive in time.</p>
 +
<p>We performed 2 different kinds of experiments, verifying both antimicrobial expressing part McbABCD and immunity part McbEFG, by testing the effect MccB17 may exert on the metabolism and survival state of microbes they co-cultured with.</p>
 +
</div>
  
−
  <h4>Safety training</h4>
+
<div class="clear"></div><!-- 一个章节结束需要clear来留白 -->
  
−
  <p>All members of our team have taken online courses in biosafety, such as hazardous chemicals, laboratory waste, special equipment safety, accident handling, and passed the laboratory safety examination required by the Assets and Security Department of our university before we entering the lab.</p>
+
<h4>Influences on metabolism</h4>
 +
  <div class="column two_thirds_size">
 +
  <img src="https://static.igem.org/mediawiki/2020/d/d5/T--Fudan--img_flowchart1.svg" alt="flowchart IPTG" />
 +
  <h6>Figure 1. Flowchart of the IPTG induction experiments</h6>
 +
  </div>
 +
  <div class="column third_size">
 +
    <p>We cultured WT (<i>E. coli</i> expressing GFP driven by Plac), PxAD (<i>E. coli</i> expressing <i>mcbABCD</i> driven by P1\2\9\11\12\13) and Px (<i>E. coli</i> expressing mcbABCD-mcbEFG-PtetR driven by P1\2\9\11\12\13) separately with similar OD.
 +
</p><p>
 +
When the OD of WT reaches 0.6, we mixed WT with PxAD or Px, with ratio of 1:1, 4:1, 10:1, then induce them with IPTG for 2 hours. Here, the control group would be merely WT with IPTG and WT without IPTG.
 +
</p><p>
 +
After mixture, we immediately put the starting cultures on ice to cease the proliferation and measure their OD for a precise mixture ratio. Thus, the ratio in the graph down below has been adjusted to be more accurate in the ratio of cell numbers in the mixture (different from the ratio on the flowchart). Finally, all samples were measured by a plate reader.
 +
</p>
 +
  </div>
 +
  <div class="clear"></div><!-- 一个章节结束需要clear来留白 -->
  
−
<h4>Lab work</h4>
+
<h4>Results from IPTG induction experiments</h4>
 +
  <div class="column third_size">
 +
  <img src="https://static.igem.org/mediawiki/2020/1/16/T--Fudan--5mix7.jpeg" alt="results IPTG 5 7" />
 +
<h6>Figure 2. MEFL/particle for different Px and PxAD with the ratio of mixure WT:Px=5:7</h6>
 +
  </div>
 +
<div class="column third_size">
 +
<img src="https://static.igem.org/mediawiki/2020/b/b6/T--Fudan--20mix7.jpeg" alt="results IPTG 20 7" />
 +
<h6>Figure 3. MEFL/particle for different Px and PxAD with the ratio of mixure WT:Px=20:7</h6>
 +
  </div>
 +
<div class="column third_size">
 +
<img src="https://static.igem.org/mediawiki/2020/8/89/T--Fudan--50mix7.jpeg" alt="results IPTG 50 7" />
 +
<h6>Figure 4. MEFL/particle for different Px and PxAD with the ratio of mixure WT:Px=50:7</h6>
 +
  </div>
  
−
   <p>Our experiments were all performed in the laboratory of Prof. Liang Cai in the School of Life Sciences, which is qualified for Biosafety Level 2. For safety equipment, the lab has biosafety cabinets allowing us to carry out most experiments safely. There is a cabinet to store the hazardous reagents, the key of which only Prof. Cai has, and have a dedicated refrigerator for iGEM to store our experimental materials including plasmids and primers. Fire extinguishers, fire hydrants, emergency spray, and other safety facilities are also available in the lab to deal with different accidental situations.</p>
+
   <div class="column full_size">
 +
<p>Our data has been calibrated with the Excel data analysis template provided by the 2019 iGEM Measurement Committee, where MEFL stands for the absolute units of Fluorescent Intensity for Green Fluorescent proteins and particle stands for the absolute units of cell count, so that our Y axis represents fluorescent intensity per cell under iGEM standard.
 +
</p>
 +
<p>We could see an overall decrease of GFP expression in nearly all groups co-cultured with Px or PxAD than the control group which only contains WT, indicating that the antimicrobial peptide (MccB17) encoded by <i>mcbABCD</i> does have a negative effect on microbial. In this case, the living status of WT cells and its function in creating GFP is strongly restricted by the unsuitable environment, proving that our part <i>mcbABCD</i> works as designed.
 +
</p>
 +
<p>While inside each paired group, there clearly are a better inhibition effect in Px group than PxAD group, as the MEFL/particle is much lower in the Px group than the PxAD group when driven by certain promoters. This shows that the immunity function of McbEFG is working successfully, as the E. coli expressing <i>mcbEFG</i> can help the engineered strain to survive with efflux exporting the toxic peptide, as well as better killing off other strains to gain survival advantage.
 +
</p>
  
−
<p>As the supervisor of the lab, Prof. Cai also provided us with systematic safety guidelines and in-field training before we started the lab affair. Every time we conducted our experiments, at least one senior student was present overseeing our daily experimental operations following all the guidelines. Some of the guidelines were as follows: </p>
+
<p>Among the three graphs, it is also obvious the system works better when the McbABCD population occupies a larger percentage. When the ratio of WT:Px meets 50:7, nealy all of the groups shows very weak competitiveness or even no survival advantage at all (as in the case of P1), indicating the need of a certain amount of McbABCD population to get the system working efficiently. This result also affirms it is vital to have the antimicrobial system in our design to gain enough survival advantage for our engineered <i>E. coli</i> to colony in human intestine and further express the desired product.
 +
</p>
 +
<p>Due to the difference in strength of these promoters (P1 to P14) driven the expression of <i>mcbABCD</i>, our initial test suggested the most suitable promoter P2 for the system.
 +
</div>
  
−
                <ol>
+
<div class="clear"></div><!-- 一个章节结束需要clear来留白 -->
−
                    <li>The experimental bench and the rest of the area should be strictly separated. Everyone performing experiments must wear Lab -gown and nitrile gloves before getting into the experimental area. Masks should also be wearing to prevent <i>E. coli</i> from being taken in. Lab -gown, gloves and any other used personal protective equipment must not be taken out of the experimental area.</li>
+
−
                    <li> Experiments using hazardous chemicals like Ethidium Bromide should be conducted on a special bench. While conducting, experimenters must wear an additional pair of nitrile gloves to protect themselves and avoid contaminating the inner gloves.</li>
+
−
                    <li>If wanting to use the high-handed sterilization pan, students should ask the supervisor or other senior students to operate instead. </li>
+
−
                    <li>The last person to leave the lab should make sure that water, electricity, and the air conditioner are shut down, and lock all the doors and windows before leaving.</li>
+
−
                </ol>
+
−
 
+
−
  <h4>Avoid contamination</h4>
+
−
  <p>All kinds of wastes were disposed of following the laboratory waste disposal policy strictly. Normal laboratory wastes are collected and emptied regularly. Contaminative and toxic waste was separately stored according to their chemical properties waiting for specialists to treat. All containers in contact with bacteria were sterilized before and after use. Experiments that might cause bacteria contamination are performed in Biosafety cabinets. Our culture medium and plates were sealed and preserved in separate refrigerators. Besides, our genetic materials were provided directly from Prof. Cai’s lab or ordered from a local biotechnology company, so there was no transfer or shipment problem involved. We have always transferred our information and experimental data with great deliberation.</p>
+
  
 +
<h4>Influnces on survival</h4>
 +
<div class="column two_thirds_size">
 +
<img src="https://static.igem.org/mediawiki/2020/6/61/T--Fudan--flowchart_kanamp.svg" alt="flowchart time-course" />
 +
<h6>Figure 5. Flowchart of the survial (time-course) experiments</h6>
 
   </div>
 
   </div>
−
  <div class="clear"></div><!-- 一个章节结束需要clear来留白 -->
 
  
−
  <h2>Project design</h2>
+
<div class="column third_size">
−
  <div class="column full_size" >
+
    <p>We cultured WT (<i>E. coli</i> expressing RFP driven by a constitutive promoter), PxAD (<i>E. coli</i> expressing <i>mcbABCD</i> driven by P1\2\9\11\12\13) and Px (<i>E. coli</i> expressing mcbABCD-mcbEFG-PtetR driven by P1\2\9\11\12\13) separately at the same time.</p>
−
  <p>Most of the organisms and parts we have chosen were widely used and we made throughout safety assessment on them before finally decided to use. And we also introduced several devices to guarantee the safety of both users and the environment.</p>
+
    <p>When the OD of WT reaches 0.6, we mixed WT with PxAD or Px, with ratio of 1:1, 4:1. Later, we collect samples from the mixture at different time points, to be measured by a plate reader.</p>
 +
    <p>Meanwhile, we plate the mixture onto plates with different antibiotics (since WT and Px/PxAD possess different antibiotic resistance) to measure the precise ratio of mixture.</p>
 
   </div>
 
   </div>
 
   <div class="clear"></div><!-- 一个章节结束需要clear来留白 -->
 
   <div class="clear"></div><!-- 一个章节结束需要clear来留白 -->
  
−
   <div class="column full_size" >
+
   <h4>Results from survial experiments</h4>
  
−
   <h4>Nissle 1917</h4>
+
<div class="row">
−
   <p>We chose <i>E. coli</i> Nissle 1917 as the main chassis to take effect in our project. <i>E. coli</i> Nissle 1917 has a history of 100 years as a licensed medicinal probiotic and is one of the most intensively investigated bacterial strain used in a probiotic preparation today. Its biosafety and effect have since been extensively shown in numerous trials<sup>[1]</sup>. Neither the gene of prominent virulence or the production of toxins have been found in Nissle 1917 and this strain also has proven to lack immunotoxic properties<sup>[2]</sup>. What’s more, Nissle has the ability to express several fitness factors, such as microcins, adhesins and iron uptake systems that facilitate their colonization within the host<sup>[3]</sup>. In addition, Nissle is in Group 1 and has been handled properly in the Safety Level 2 laboratory. To summarize, Nissle 1917 proves to be the perfect chassis for our project. Unfortunately, due to the limited time, we only verified the designed polypeptide chain can be expressed and function normally in <i>E. coli</i>. We hoped to consummate experiments Nissle 1917 in the future.</p>
+
   <div class="col m2 l3 hide-on-small-only">&#160;</div>
 +
   <div class="col s12 m8 l6">
 +
    <img src="https://static.igem.org/mediawiki/2020/b/b9/T--Fudan--AUP21.jpeg" alt="time-course results" style="width:100%" />
 +
<h6>Figure 6. Calculated Bacteria Counts with the ratio of mixure Px/PxAD:WT=1:1</h6>
 +
    </div>
 +
</div>
  
−
  <h4><i>E. coli</i></h4>
+
  <div class="column full_size">
 +
<p> Our data has been calibrated with the Excel data analysis template provided by the 2019 iGEM Measurement Committee. In this chart, Calculated Bacteria Counts stands for the relative increase of OD, which represents the total number of cells, and MERL, which represents the total number of WT cells. As we have screened out P2 in front, here we used the mixture ratio of P2(P2AD):WT=1:1 to find out more detailed effect that antimicrobial peptide expressing system can exert.</p>
 +
<p>We could see the population increase are not significant in total over time, yet WT grows rather fast at first in the P2AD group, indicating its lack of immunity part McbEFG can lead to the death of engineered <i>E. coli</i>. Thus shows less restrain on the WT cells. Yet, as MccB17 accumulates, a clear decrease can be observed when time extends in both P2 and P2AD, suggesting our antimicrobial peptide expressing system is working to help the engineered <i>E. coli</i> gain better survival advantage.
 +
</p>
 +
  </div>
 +
  <div class="clear"></div><!-- 一个章节结束需要clear来留白 -->
  
−
  <p><i>E. coli</i> strain DH5α and BL21 have been used to carry out molecular cloning and obtain the designed plasmids in our project. They are non-pathogenic and can hardly survive in the natural environment. Both strains are in Group 1 and have been handled properly in the Safety Level 2 laboratory.</p>
+
<h2>Reduced leakage of PtetR (improved part)</h2>
 +
<div class="column full_size">
 +
    <p>We have measured the expression level of PtetO2 (<a href="http://parts.igem.org/Part:BBa_K3606006" target="_blank">BBa_K3606006</a>), PtetO3 (<a href="http://parts.igem.org/Part:BBa_K3606007" target="_blank">BBa_K3606007</a>) along with the original PtetR (<a href="http://parts.igem.org/Part:BBa_R0040" target="_blank">BBa_R0040</a>) via plate reader by combining them with GFP, when they were induced by different concentration of anhydrotetracycline (aTc) or not induced.</p>
 +
  </div>
  
−
   <h4>Kill Switch</h4>
+
   <div class="column half_size">
 +
    <img src="https://static.igem.org/mediawiki/2020/5/51/T--Fudan--aTc_0.jpg" alt="figure 1" />
 +
    <h6>Figure 7. When aTc=0, the expression level of the promoter and bacterial OD: When OD>0.6, the promoter data is meaningful.</h6>
  
−
  <p>Although our project remained at an experimental stage, no usage outside the lab or clinical trials were applied, we devised some approaches to avoid potential problems. In order to ensure our engineered probiotic will not jeopardize the environment or users, this year, we integrated two kinds of Kill Switches into our project.</p>
+
    <img src="https://static.igem.org/mediawiki/2020/d/d3/T--Fudan--aTc_200.jpg" alt="figure 3" />
−
<p>To deprive of the survivability of engineered Nissle in the environment when excreted from the human intestine, we introduced the first Kill Switch. The first Kill Switch is a cold triggered Kill Switch, consisting of a toxin/antitoxin system as a major functional part and an RNA thermometer to regulate it. (Check to <a href="/Team:Fudan/Design#kill_switch">see the detailed design</a> and our consideration in<a href="/Team:Fudan/Engineering"> engineering</a>.)  And bacteria escaped from Kill Switch could be eliminated by the complementary automatic toilet cleaner.</p>
+
    <h6>Figure 9. When aTc=200, the expression level of the promoter and bacterial OD: When OD>0.6, the promoter data is meaningful.</h6>
−
</div>
+
  </div>
  
 
   <div class="column half_size">
 
   <div class="column half_size">
−
     <img src="https://static.igem.org/mediawiki/2020/2/25/T--Fudan--img_ks4.svg" alt="cold triggered MazF/MazE Kill Switch under body temperature(37℃)" />
+
     <img src="https://static.igem.org/mediawiki/2020/0/06/T--Fudan--aTc_100.jpg" alt="figure 2" />
 +
    <h6>Figure 8. When aTc=100, the expression level of the promoter and bacterial OD: When OD>0.6, the promoter data is meaningful.</h6>
 +
    <img src="https://static.igem.org/mediawiki/2020/4/46/T--Fudan--aTc_400.jpg" alt="figure 4" />
 +
    <h6>Figure 10. When aTc=400, the expression level of the promoter and bacterial OD: When OD>0.6, the promoter data is meaningful.</h6>
 
   </div>
 
   </div>
−
   <div class="column half_size">
+
 
−
     <img src="https://static.igem.org/mediawiki/2020/a/a4/T--Fudan--img_ks3.svg" alt="cold triggered MazF/MazE Kill Switch outside the body(30℃)" />
+
   <div class="column full_size">
 +
     <p>Under the induction of various concentrations of aTc, the expression level of PtetO2 is very low, but the expression level of PtetO3 is moderate. Although the expression level of PtetO3 is lower than PtetR, it is very suitable for our project design. Since PtetO3 is used to drive McbEFG, if McbEFG translates too much channel protein, then when McbEFG is turned off, the expressed channel protein will take a long time to degrade completely, reducing the sensitivity of quorum sensing.</p>
 
   </div>
 
   </div>
  
−
  <div class="column full_size" >
+
<div class="clear"></div>
−
<h6>Figure 1. Cold triggered MazF/MazE Kill Switch under body temperature (37℃) and outside the body (30℃).</h6></div>
+
  
−
<div class="column full_size" >
+
<div class="column third_size">
−
  <p>The second Kill Switch is an L-arabinose-activated promoter followed by a translational inhibitor MazF made by 19BNU_China (<a target="_blank" href="http://parts.igem.org/Part:BBa_K3036005">BBa_K3036005</a>). It is introduced to enable users to eliminate the engineered bacteria at will, meanwhile, it won’t upset native intestinal flora. Whenever users want to stop using our probiotic, they can intake an L-arabinose tablet which has easy access from a health food store. </p>
+
    <img src="https://static.igem.org/mediawiki/parts/7/7a/T--Fudan--rate_100.jpg" alt="figure 5">
 +
    <h6>Figure 11. When aTc=100, fold of increase of promoters</h6>
 +
    </div>
 +
<div class="column third_size">
 +
    <img src="https://static.igem.org/mediawiki/2020/a/a4/T--Fudan--rate_200.jpg" alt="figure 6">
 +
    <h6>Figure 12. When aTc=200, fold of increase of promoters</h6>
 +
    </div>
 +
<div class="column third_size">
 +
    <img src="https://static.igem.org/mediawiki/2020/0/0f/T--Fudan--rate_400.jpg" alt="figure 7">
 +
    <h6>Figure 13. When aTc=400, fold of increase of promoters</h6>
 +
    </div>
  
−
  <h4>Future expectation</h4>
+
<div class="clear"></div>
  
−
  <p>Antibiotics resistance gene present a safety concern and is forbidden in probiotic consumption. However, it has been paid relatively less attention by previous iGEM teams working on probiotics. Antibiotics resistance gene is the most commonly used Selective marker gene. While encoded on the plasmid, it has the possibility to transfer to intestinal flora in the host and hurries the development of antibiotic-resistant strains<sup>[4]</sup>. Our project remains at an experimental stage that won’t implement experiments in vivo and we took the huge expenditures of both cost and time into consideration, we still used antibiotics resistance genes to make screenings.</p>
+
<div class="column full_size">
−
<p>For a commercialization plan in the future, we may try recombinase-mediated DNA insertion (RMDI) to replace antibiotics resistance genes to screen<sup>[5]</sup> or insert our genetic circuits into the genome to inhibit the horizontal transfer directly.</p>
+
    <p>The results show that among the three promoters, PtetO3 has the highest fold of increase. This means that it is the most sensitive to the induction of aTc, which can improve the sensitivity of quorum sensing.
−
</div>
+
</p>
 +
  </div>
  
−
<div class="clear"></div>
+
<div class="clear"></div><!-- 一个章节结束需要clear来留白 -->
  
−
  <h2>Safety during the COVID-19</h2>
+
<h2>Demonstration of CaAP and signal-peptide guided CaAP secretion system</h2>
 +
<div class="column full_size">
 +
<h4>CaAP (Calcium Absorption Peptide)</h4>
 +
<p>We hope that our project can solve the problem of calcium deficiency in the elderly. According to our investigation, due to the diet structure, the calcium absorption of Chinese people is not enough, so we <a href="/Team:Fudan/Design">design</a> CaAP from the perspective of improving the calcium absorption rate of the elderly.</p>
  
−
  <div class="column full_size">
+
<p>We have successfully cloned our designed CaAP (Calcium Absorption Peptide) into pFAB plasmid and transformed the construct into <i>E. coli</i> BL21 strain to demonstrate its expression.</p>
−
  <h4>Project implementation during the pandemic</h4>
+
</div>
  
−
  <p>Given the outbreak of the COVID-19 pandemic, we made extra efforts to protect ourselves and our communities. We strictly complied with all guidance from our university and local government the whole time. The first thing we ought to consider was to guarantee team members’ own health and safety. On account of no admittance to campus by the university, we converted our meetings to online remote conferences. Members that got back to school to carry out wet lab experiments have obtained the permission of the university. Though our university is located in a very low-risk area, we wore masks carefully whenever in public occasions to prevent infection. In the meantime, the physical condition of every team member was closely monitored by the “Hello Fudan” applet.</p>
 
  
−
   <h4>HP and education during pandemic</h4>
+
<div class="column half_size">
−
  <p>During this particular time, we especially emphasize social distancing and focus on online interactions. In integrated human practice, we used an online questionnaire to investigate the public about osteoporosis awareness and calcium absorption. To better access the old, our target group, considering their weakness and lack of internet skills, we guide the young to help the aged in their family finish it. Besides, we use e-mails to connect with the osteoporosis field clinician and relevant professors, changing face-to-face interviews with the WeChat voice call. Aiming to engage current osteoporosis issues, we investigated published surveys, combining them with the 2018 China guideline for diagnosis and treatment of senile osteoporosis to analyze the current situation. We cooperate with Tianjin university Team for education, creating an online life science summer camp for middle school students. To avoid public education events, we design an online audio course and textbook to introduce basic biology knowledge.</p>
+
   <h4> Secretion Peptides guided CaAP</h4>
 +
<p>To promote calcium bioavailability, CaAP needs to be expressed in our probiotics and subsequently secreted into the extracellular space. Therefore, we linked 5 signal peptides which belong to the Type II secretion system in <i>E. coli</i>, to the N terminal of CaAP, respectively.
 +
The 5 secretion peptides are listed below:</p>
 +
<table>
 +
<tr>
 +
<td> NSP4 </td> <td> <a href="http://parts.igem.org/Part:BBa_K3606042" target="_blank">BBa_K3606042</a> </td>
 +
</tr>
 +
<tr>
 +
<td> OmpA </td> <td> <a href="http://parts.igem.org/Part:BBa_K3606043" target="_blank">BBa_K3606043</a> </td>
 +
</tr>
 +
<tr>
 +
<td> DsbA </td> <td> <a href="http://parts.igem.org/Part:BBa_K3606030" target="_blank">BBa_K3606030</a> </td>
 +
</tr>
 +
<tr>
 +
<td> PelB </td> <td> <a href="http://parts.igem.org/Part:BBa_K208004" target="_blank">BBa_K208004</a> </td>
 +
</tr>
 +
<tr>
 +
<td> PhoA </td> <td> <a href="http://parts.igem.org/Part:BBa_K808028" target="_blank">BBa_K808028</a> </td>
 +
</tr>
 +
</table>
 
</div>
 
</div>
  
−
<div class="clear"></div><!-- 一个章节结束需要clear来留白 -->
+
<div class="column half_size">
 +
    <p>To test the secretion efficiency of our 5 secretion peptides and compare their secretion capabilities, we also fused them to MBP (Maltose Binding Protein) that normally expressed in the cytoplasm of <i>E. coli</i> cells. Driven by an IPTG inducible promoter Ptac, we can achieve quantified characterization of these secretion peptides through differences of the amount of signal-peptide-MBP fusion protein in bacterial lysate and culture medium.</p>
 +
    <p>We have successfully cloned OmpA guided MBP (Maltose Binding Protein) and expressed it in <i>E. coli</i> BL21. As shown in Figure 14,the amount of MBP in the lysate shows no difference with or without IPTG induction, suggesting that OmpA has the strong ability to secrete MBP into the extracellular space. As for the relatively low amount of MBP in the culture medium concentrated by TCA than in the cell lysate (shown in Figure 15), it may be caused by other interference factors during the preparation of protein samples.</p>
 +
</div>
  
−
  <div class="column full_size"><div class="highlight decoration_background decoration_B_full">
+
<div class="column half_size">
−
     <p>We have paid high attention to safety issues on project design, project implementation and health precaution during the COVID-19 pandemic following the instruction of the Safety and Security Community. Our project was accomplished successfully without bringing potential risks to ourselves, our communities and the environment.</p>
+
<div class="row">
−
  </div></div>
+
  <div class="col s2 m3 l2"></div>
 +
  <div class="col s8 m6 l6">
 +
     <img src="https://static.igem.org/mediawiki/2020/0/05/T--Fudan--denoted_gel2_OmpA_MBP.png" alt="OmpA MBP lysate" style="width:75%"/>
 +
    <h6>Figure 14. OmpA-MBP expression with and without IPTG induction in the bacterial lysate.</h6>
 +
    </div>
 +
</div>
 +
</div>
 +
<div class="column half_size">
 +
<div class="row">
 +
  <div class="col s2 m3 l2"></div>
 +
  <div class="col s8 m6 l6">
 +
    <img src="https://static.igem.org/mediawiki/2020/3/3b/T--Fudan--denoted_gel3_OmpA_MBP.png" alt="1_1" style="width:100%"/>
 +
    <h6>Figure 15. OmpA-MBP expression with and without IPTG induction in the culture medium concentrated by TCA.</h6>
 +
    </div>
 +
</div>
 +
</div>
 +
<div class="column full_size">
 +
    <p>To test the secretion efficiency of our 5 secretion peptides fused CaAP (Calcium Absorption Peptide) guided by 5 secretion peptides, we cloned the Secretion peptide-CaAP segment into the pGEX vector respectively and transformed into <i>E. coli</i> BL21 strain. Further, quantified characterization of these secretion peptides fused with CaAP is expected to be achieved through the differences of calcium ion concentration in bacterial lysate and culture medium.</p>
 +
</div>
  
−
<div class="clear"></div><!-- 一个章节结束需要clear来留白 -->
+
<div class="row">
−
<h2>Reference</h2>
+
  <div class="col m2 l3 hide-on-small-only">&#160;</div>
−
   <div class="column full_size">
+
   <div class="col s12 m8 l6">
−
                    <p>[1] Ulrich Sonnenborn, <i>Escherichia coli</i> strain Nissle 1917—from bench to bedside and back: history of a special <i>Escherichia coli</i> strain with probiotic properties, FEMS Microbiology Letters, Volume 363, Issue 19, October 2016, fnw212, https://doi.org/10.1093/femsle/fnw212 <br/>
+
    <img src="https://static.igem.org/mediawiki/parts/6/67/T--Fudan--denoted_gel2_signalpeptides.png" alt="2_6" style="width:100%">
−
                    [2] Behnsen, Judith et al. “Probiotics: properties, examples, and specific applications.” Cold Spring Harbor perspectives in medicine vol. 3,3 a010074. 1 Mar. 2013, doi:10.1101/cshperspect.a010074<br/>
+
<h6>Figure 16. NSP4/PhoA/OmpA-CaAP expression with and without IPTG induction in the bacterial lysate.</h6>
−
                    [3] Yu, Xiaoli et al. “Bioengineered <i>Escherichia coli</i> Nissle 1917 for tumour-targeting therapy.” Microbial biotechnology vol. 13,3 (2020): 629-636. doi:10.1111/1751-7915.13523<br/>
+
    </div>
 +
</div>
  
−
                    [4] de Melo Pereira, G.V., et al., How to select a probiotic? A review and update of methods and criteria. Biotechnology Advances, 2018. 36(8): p. 2060-2076.<br/>
+
<div class="row">
−
                    [5] Turan, S., et al., Recombinase-Mediated Cassette Exchange (RMCE): Traditional Concepts and Current Challenges. Journal of Molecular Biology, 2011. 407(2): p. 193-221.</p>
+
  <div class="col m2 l3 hide-on-small-only">&#160;</div>
 +
  <div class="col s12 m8 l6">
 +
    <img src="https://static.igem.org/mediawiki/parts/0/0d/T--Fudan--File-T--Fudan--denoted_gel3_signalpeptides.png" alt="2_6" style="width:100%">
 +
<h6>Figure 17. NSP4/PhoA/OmpA-CaAP expression expression with and without IPTG induction in the culture medium concentrated by trichloroacetic acid (TCA) precipitation.</h6>
 +
    </div>
 
</div>
 
</div>
−
<div class="clear"></div><!-- 一个章节结束需要clear来留白 -->
 
  
−
<div class="column full_size"><div id="FudanSignature">
+
<div class="column full_size">
−
    <p>Signature: <a href="/Team:Fudan/Attributions#ZhangJQ" class="z-depth-2">Jingqi</a>
+
    <p>Please note that our modeling results are at <a href="/Team:Fudan/Model">/Team:Fudan/Model</a>, and our online survey results are at <a href="/Team:Fudan/Human_Practices">/Team:Fudan/Human_Practices</a>.</p>
−
      </p>
+
</div>
−
</div></div>
+
 
 +
  <div class="clear"></div><!-- 一个章节结束需要clear来留白 -->
 +
 
 +
  <div class="column full_size"><div id="FudanSignature">
 +
      <p>Signature:
 +
                    <a href="/Team:Fudan/Attributions#ZhangZR" class="z-depth-2">Zhenru</a>
 +
                    <a href="/Team:Fudan/Attributions#ZhangGC" class="z-depth-2">Gaochen</a>
 +
        </p>
 +
  </div></div>
  
 
<!-- 此处以上可更改
 
<!-- 此处以上可更改

Latest revision as of 02:39, 2 December 2020
 
results

Improvement of the antimicrobial peptide expressing system

Here, we tried to improve the former antimicrobial peptide (Mccb17) expression, based on 2019 Fudan BBa_K3245010. By dividing into the peptide expressing part and the immunity part, we firstly tested whether the polycistron could work properly and separately, then tried to manipulate each of their expression levels for better results.

Verification of the expression

First, to affirm the system could work properly, we started from the very beginning by testing the expression of each mcb gene in the cluster as well as of the polycistrons in each part. We constructed 10 plasmids (with pGEX backbone adapted to perform IPTG induced expression), containing mcbA/ mcbB/ mcbC/ mcbD/ mcbE/ mcbF/ mcbG/ mcbABCD/ mcbBCD/ mcbBC respectively, transformed successfully them into E. coli BL21. We have made further confirmation that every part can be induced successfully due to the correct bands on the SDS-PAGE gels, only shown in IPTG induced samples. If needed, we could purify these protein products via immobilized Ni affinity chromatography (GST was replaced with His-tag in our constructs).

Verification of the function

As our design page describes, McbABCD should show a certain degree of restrain to both the growth and metabolism of its co-cultured cells. While with McbEFG, engineered E. coli are supposed to have better immunity themselves against the MccB17 coded by mcbABCD, so as to gain advantage, affect the surrounding WT’s metabolism (expressing of GFP in our experiments), and better survive in time.

We performed 2 different kinds of experiments, verifying both antimicrobial expressing part McbABCD and immunity part McbEFG, by testing the effect MccB17 may exert on the metabolism and survival state of microbes they co-cultured with.

Influences on metabolism

flowchart IPTG
Figure 1. Flowchart of the IPTG induction experiments

We cultured WT (E. coli expressing GFP driven by Plac), PxAD (E. coli expressing mcbABCD driven by P1\2\9\11\12\13) and Px (E. coli expressing mcbABCD-mcbEFG-PtetR driven by P1\2\9\11\12\13) separately with similar OD.

When the OD of WT reaches 0.6, we mixed WT with PxAD or Px, with ratio of 1:1, 4:1, 10:1, then induce them with IPTG for 2 hours. Here, the control group would be merely WT with IPTG and WT without IPTG.

After mixture, we immediately put the starting cultures on ice to cease the proliferation and measure their OD for a precise mixture ratio. Thus, the ratio in the graph down below has been adjusted to be more accurate in the ratio of cell numbers in the mixture (different from the ratio on the flowchart). Finally, all samples were measured by a plate reader.

Results from IPTG induction experiments

results IPTG 5 7
Figure 2. MEFL/particle for different Px and PxAD with the ratio of mixure WT:Px=5:7
results IPTG 20 7
Figure 3. MEFL/particle for different Px and PxAD with the ratio of mixure WT:Px=20:7
results IPTG 50 7
Figure 4. MEFL/particle for different Px and PxAD with the ratio of mixure WT:Px=50:7

Our data has been calibrated with the Excel data analysis template provided by the 2019 iGEM Measurement Committee, where MEFL stands for the absolute units of Fluorescent Intensity for Green Fluorescent proteins and particle stands for the absolute units of cell count, so that our Y axis represents fluorescent intensity per cell under iGEM standard.

We could see an overall decrease of GFP expression in nearly all groups co-cultured with Px or PxAD than the control group which only contains WT, indicating that the antimicrobial peptide (MccB17) encoded by mcbABCD does have a negative effect on microbial. In this case, the living status of WT cells and its function in creating GFP is strongly restricted by the unsuitable environment, proving that our part mcbABCD works as designed.

While inside each paired group, there clearly are a better inhibition effect in Px group than PxAD group, as the MEFL/particle is much lower in the Px group than the PxAD group when driven by certain promoters. This shows that the immunity function of McbEFG is working successfully, as the E. coli expressing mcbEFG can help the engineered strain to survive with efflux exporting the toxic peptide, as well as better killing off other strains to gain survival advantage.

Among the three graphs, it is also obvious the system works better when the McbABCD population occupies a larger percentage. When the ratio of WT:Px meets 50:7, nealy all of the groups shows very weak competitiveness or even no survival advantage at all (as in the case of P1), indicating the need of a certain amount of McbABCD population to get the system working efficiently. This result also affirms it is vital to have the antimicrobial system in our design to gain enough survival advantage for our engineered E. coli to colony in human intestine and further express the desired product.

Due to the difference in strength of these promoters (P1 to P14) driven the expression of mcbABCD, our initial test suggested the most suitable promoter P2 for the system.

Influnces on survival

flowchart time-course
Figure 5. Flowchart of the survial (time-course) experiments

We cultured WT (E. coli expressing RFP driven by a constitutive promoter), PxAD (E. coli expressing mcbABCD driven by P1\2\9\11\12\13) and Px (E. coli expressing mcbABCD-mcbEFG-PtetR driven by P1\2\9\11\12\13) separately at the same time.

When the OD of WT reaches 0.6, we mixed WT with PxAD or Px, with ratio of 1:1, 4:1. Later, we collect samples from the mixture at different time points, to be measured by a plate reader.

Meanwhile, we plate the mixture onto plates with different antibiotics (since WT and Px/PxAD possess different antibiotic resistance) to measure the precise ratio of mixture.

Results from survial experiments

 
time-course results
Figure 6. Calculated Bacteria Counts with the ratio of mixure Px/PxAD:WT=1:1

Our data has been calibrated with the Excel data analysis template provided by the 2019 iGEM Measurement Committee. In this chart, Calculated Bacteria Counts stands for the relative increase of OD, which represents the total number of cells, and MERL, which represents the total number of WT cells. As we have screened out P2 in front, here we used the mixture ratio of P2(P2AD):WT=1:1 to find out more detailed effect that antimicrobial peptide expressing system can exert.

We could see the population increase are not significant in total over time, yet WT grows rather fast at first in the P2AD group, indicating its lack of immunity part McbEFG can lead to the death of engineered E. coli. Thus shows less restrain on the WT cells. Yet, as MccB17 accumulates, a clear decrease can be observed when time extends in both P2 and P2AD, suggesting our antimicrobial peptide expressing system is working to help the engineered E. coli gain better survival advantage.

Reduced leakage of PtetR (improved part)

We have measured the expression level of PtetO2 (BBa_K3606006), PtetO3 (BBa_K3606007) along with the original PtetR (BBa_R0040) via plate reader by combining them with GFP, when they were induced by different concentration of anhydrotetracycline (aTc) or not induced.

figure 1
Figure 7. When aTc=0, the expression level of the promoter and bacterial OD: When OD>0.6, the promoter data is meaningful.
figure 3
Figure 9. When aTc=200, the expression level of the promoter and bacterial OD: When OD>0.6, the promoter data is meaningful.
figure 2
Figure 8. When aTc=100, the expression level of the promoter and bacterial OD: When OD>0.6, the promoter data is meaningful.
figure 4
Figure 10. When aTc=400, the expression level of the promoter and bacterial OD: When OD>0.6, the promoter data is meaningful.

Under the induction of various concentrations of aTc, the expression level of PtetO2 is very low, but the expression level of PtetO3 is moderate. Although the expression level of PtetO3 is lower than PtetR, it is very suitable for our project design. Since PtetO3 is used to drive McbEFG, if McbEFG translates too much channel protein, then when McbEFG is turned off, the expressed channel protein will take a long time to degrade completely, reducing the sensitivity of quorum sensing.

figure 5
Figure 11. When aTc=100, fold of increase of promoters
figure 6
Figure 12. When aTc=200, fold of increase of promoters
figure 7
Figure 13. When aTc=400, fold of increase of promoters

The results show that among the three promoters, PtetO3 has the highest fold of increase. This means that it is the most sensitive to the induction of aTc, which can improve the sensitivity of quorum sensing.

Demonstration of CaAP and signal-peptide guided CaAP secretion system

CaAP (Calcium Absorption Peptide)

We hope that our project can solve the problem of calcium deficiency in the elderly. According to our investigation, due to the diet structure, the calcium absorption of Chinese people is not enough, so we design CaAP from the perspective of improving the calcium absorption rate of the elderly.

We have successfully cloned our designed CaAP (Calcium Absorption Peptide) into pFAB plasmid and transformed the construct into E. coli BL21 strain to demonstrate its expression.

Secretion Peptides guided CaAP

To promote calcium bioavailability, CaAP needs to be expressed in our probiotics and subsequently secreted into the extracellular space. Therefore, we linked 5 signal peptides which belong to the Type II secretion system in E. coli, to the N terminal of CaAP, respectively. The 5 secretion peptides are listed below:

NSP4 BBa_K3606042
OmpA BBa_K3606043
DsbA BBa_K3606030
PelB BBa_K208004
PhoA BBa_K808028

To test the secretion efficiency of our 5 secretion peptides and compare their secretion capabilities, we also fused them to MBP (Maltose Binding Protein) that normally expressed in the cytoplasm of E. coli cells. Driven by an IPTG inducible promoter Ptac, we can achieve quantified characterization of these secretion peptides through differences of the amount of signal-peptide-MBP fusion protein in bacterial lysate and culture medium.

We have successfully cloned OmpA guided MBP (Maltose Binding Protein) and expressed it in E. coli BL21. As shown in Figure 14,the amount of MBP in the lysate shows no difference with or without IPTG induction, suggesting that OmpA has the strong ability to secrete MBP into the extracellular space. As for the relatively low amount of MBP in the culture medium concentrated by TCA than in the cell lysate (shown in Figure 15), it may be caused by other interference factors during the preparation of protein samples.

OmpA MBP lysate
Figure 14. OmpA-MBP expression with and without IPTG induction in the bacterial lysate.
1_1
Figure 15. OmpA-MBP expression with and without IPTG induction in the culture medium concentrated by TCA.

To test the secretion efficiency of our 5 secretion peptides fused CaAP (Calcium Absorption Peptide) guided by 5 secretion peptides, we cloned the Secretion peptide-CaAP segment into the pGEX vector respectively and transformed into E. coli BL21 strain. Further, quantified characterization of these secretion peptides fused with CaAP is expected to be achieved through the differences of calcium ion concentration in bacterial lysate and culture medium.

 
2_6
Figure 16. NSP4/PhoA/OmpA-CaAP expression with and without IPTG induction in the bacterial lysate.
 
2_6
Figure 17. NSP4/PhoA/OmpA-CaAP expression expression with and without IPTG induction in the culture medium concentrated by trichloroacetic acid (TCA) precipitation.

Please note that our modeling results are at /Team:Fudan/Model, and our online survey results are at /Team:Fudan/Human_Practices.

Signature: Zhenru Gaochen