Difference between revisions of "Team:Fudan/Poster"

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">CaAP</span> expression.</p>
 
">CaAP</span> expression.</p>
  
<p>McbA,B,C,D jointly complete the secretion of MccB17. Among them, peptide product from <i>mcbA</i> is the precursor of the antimicrobial peptide, and McbB,C,D is responsible for the post-translational modification of McbA, to produce MccB17. McbG provides the immunity against MccB17, and McbE,F assemble the channel to export McbG. When the surrounding bacteria absorb MccB17 but not McbG, they would die.</p>
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<p>McbA,B,C,D jointly complete the secretion of MccB17. Among them, peptide product from <i>mcbA</i> is the precursor of the antimicrobial peptide, and McbB,C,D is responsible for the post-translational modification of McbA, to produce MccB17. McbE,F assemble the channel to export MccB17. McbG provides the immunity against MccB17. If the engineered bacteria express only McbA,B,C,D, but neither McbE,F nor McbG, intracellular accumulated MccB17 will kill itself. When the surrounding bacteria absorb MccB17 but not McbG, they would die.</p>
  
 
<p>We place <i>mcbABCD</i> under the control of a constitutive promoter, and use PtetR drive the expression of McbEFG. After the density of engineered bacteria increases to a certain extent, the heterdimers formed by the products of <i>luxI</i> and <i>luxR</i> will accumulate and activate the luxPR promoter.<br/>
 
<p>We place <i>mcbABCD</i> under the control of a constitutive promoter, and use PtetR drive the expression of McbEFG. After the density of engineered bacteria increases to a certain extent, the heterdimers formed by the products of <i>luxI</i> and <i>luxR</i> will accumulate and activate the luxPR promoter.<br/>
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<p>Improved from last year, we: <span class="bgMenu">(1)</span> use low-copy plasmid backbone pFAB to reduce the expression burden on engineered bacteria; <span class="bgMenu">(2)</span> low expression promoters to drive <i>mcbABCD</i>; <span class="bgMenu">(3)</span> PtetR drives <i>mcbEFG</i> from the reverse strand, to avoid translational readthrough; <span class="bgMenu">(4)</span> validate individual McbA/B/C/D/E/F/G expression in an IPTG induction setting.</p>
 
<p>Improved from last year, we: <span class="bgMenu">(1)</span> use low-copy plasmid backbone pFAB to reduce the expression burden on engineered bacteria; <span class="bgMenu">(2)</span> low expression promoters to drive <i>mcbABCD</i>; <span class="bgMenu">(3)</span> PtetR drives <i>mcbEFG</i> from the reverse strand, to avoid translational readthrough; <span class="bgMenu">(4)</span> validate individual McbA/B/C/D/E/F/G expression in an IPTG induction setting.</p>
  
<p>Further, we quantitively showed the antibacterial effect with our compositve part <a href="http://parts.igem.org/Part:BBa_K3606029" target="_blank">BBa_K3606029</a>, which is RFC21 assembly compatible.<br/>
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<p>Further, we quantitively showed the antibacterial effect with our favorite compositve part <a href="http://parts.igem.org/Part:BBa_K3606029" target="_blank">BBa_K3606029</a>, which is RFC21 assembly compatible.<br/>
<img src="https://static.igem.org/mediawiki/2020/b/bf/T--Fudan--Poster_filler.jpg" alt="to be replaced" /><br/>
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<img src="https://static.igem.org/mediawiki/2020/e/e9/T--Fudan--Poster_mcbABCD-EFG_by_IPTG1.png" alt="Mcb by IPTG1" /><br/>
In the figures above, <span class="bgMenu">MEFL</span> is the absolute value of total green fluorescence and <span class="bgMenu">particle</span> represents the absolute counts of bacteria, so that Y axis represents green fluorescence per bacteria. In those experiments, we mixed WT <i>E. coli</i> (expressing GFP driven by Plac) with mcbABCD-(mcbEFG-PtetR) at different ratios (5:7, 20:7, 50:7), and measured <span class="bgMenu">MEFL/particle</span> two hours after adding 1 mM IPTG. We used this assay to study the antibacterial effect of <span class="span_click" onclick="
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In the figures above, <span class="bgMenu">MEFL</span> is the absolute value of total green fluorescence and <span class="bgMenu">particle</span> represents the absolute counts of bacteria, so that Y axis represents green fluorescence per bacteria. In those experiments, we mixed WT <i>E. coli</i> (expressing GFP driven by Plac) with mcbABCD-(mcbEFG-PtetR) at different ratios 5:7, 20:7 and 50:7, then measured <span class="bgMenu">MEFL/particle</span> two hours after adding 1 mM IPTG. We used this assay (experimental flow chart at the bottom of this page) to study the antibacterial effect of MccB17 on protein synthesis.</p>
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<p>Similarly, we shown that our antimicrobial peptide suppresses WT bacteria growth (data shown on <a href="/Team:Fudan/Parts#Composite" target="_blank">/Team:Fudan/Parts#Composite</a> and the main page of <a href="http://parts.igem.org/Part:BBa_K3606029" target="_blank">the part in the Registry</a>).</p>
    var pp_title = $('#demo_mcbABCD > .info > .title').html();
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<p><img src="https://static.igem.org/mediawiki/2020/f/fb/T--Fudan--Poster_flowchart-IPTG.png" alt="flow chart of MccB17 IPTG experiments" />
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">MccB17</span> on protein synthesis. Similarly, we shown that our antimicrobial peptide suppresses WT bacteria growth (data shown on <a href="/Team:Fudan/Parts#Composite" target="_blank">/Team:Fudan/Parts#Composite</a>).</p>
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<p>sdsa easd easddea sdaesae<br/>
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<img src="https://static.igem.org/mediawiki/2020/b/bf/T--Fudan--Poster_filler.jpg" alt="to be replaced" />
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</p>
 
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">MccB17</span>, McbG and <span class="span_click" onclick="
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">MccB17</span> and <span class="span_click" onclick="
 
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Revision as of 06:45, 10 November 2020

© 2020 Team:Fudan/Poster

Make calcium supplement sustainable

abstract
Bone appetite

In Asia, many old people suffer from insufficient calcium intake and related diseases such as osteoporosis. Considering that traditional calcium supplementation is not efficient and user-friendly enough, we intended to develop a kind of engineered bacteria based on E. coli that can colonize in the human intestine and secrete peptides to promote calcium absorption.

We introduced the quorum sensing system to colonize bacteria in the intestine. Once the bacteria have successfully colonized the intestinal tract, the luxPR promoter will be activated and a series of short peptides that can bind to calcium and promote calcium absorption of intestinal epithelial cells will be expressed. A kill switch is added accordingly for safety.

Our project provides a novel delivery method to provide calcium supplements sustainably. And we are actively seeking commercialization possibilities to expand our project impact, to reach more population, for the elderly's good health and well-being.

what we have done
So far, what we have achieved

项目缘起,需要HP那四张关于骨质疏松的问卷图,数据不变 重画

informgram survey
bala bala bala ba la ba la bala bala bala ba la ba la bala bala bala ba la ba la bala bala bala ba la ba la bala bala bala ba la ba la bala bala bala ba la ba la bala bala bala ba la ba la

铜奖满足,但不是罗列因为评委有一个列表给奖牌打分

  • We have xxxx bala bala bala ba la ba la
  • We have yyyy bala bala bala ba la ba la
  • We have zzzz bala bala bala ba la ba la
  • We have wxcv bala bala bala ba la ba la

银奖满足,但不是罗列因为评委有一个列表给奖牌打分

  • We have xxxx bala bala bala ba la ba la
  • We have yyyy bala bala bala ba la ba la
  • We have zzzz bala bala bala ba la ba la
  • We have wxcv bala bala bala ba la ba la

金奖满足,但不是罗列因为评委有一个列表给奖牌打分

  • We have xxxx bala bala bala ba la ba la
  • We have yyyy bala bala bala ba la ba la
  • We have zzzz bala bala bala ba la ba la
  • We have wxcv bala bala bala ba la ba la

AHL production simulation
RA counts simulation

总结的话

sdg3
SDG 3: GOOD HEALTH AND WELL-BEING

Ensure healthy lives and promote well-being for all at all ages.

Our project aims to provide a holistic solution to the problem of osteoporosis among seniors. Our online general biology course Shengyin and senior-friendly textbooks, were designed to educate the elderly on the importance of bone health.
to be replaced

Since post-menopausal women are more likely to suffer osteoporosis, we paid special attention to them. Chinese women favor traditional art crosstalk, and we combined interesting art with basic synthetic biology knowledge. When we went to the nursing house to visit the elderly, we performed it for them, and they all laughed and enjoyed our performance very much.

Another thing we did is to promote a traditional Chinese sport - Mu'lan'quan to them. Many older women liked the sport because it is both elegant and healthy. The movements of Mu'lan'quan combine dancing and Kungfu and is preferred by many females. It is convenient to practice it - anybody can do it with a fan. We hoped our effect could help the elderly women to strengthen their health and prevent osteoporosis.
to be replaced

Members of all age groups are stakeholders of osteoporosis prevention and osteoporosis treatment. Seniors are an integral part of our society, and it is our responsibility to ensure their health and quality of life.

sdg4
SDG 4: QUALITY EDUCATION

Ensure inclusive and equitable quality education and promote lifelong learning opportunities for all.

With the rapid development of technology and society, the older generation is gradually lagging behind. The elderly is facing fraud, misinformation, and diseases. Lifelong education has become an urgent need for them. We dedicated ourselves to exploring education approaches, trying to bring high-quality education for the elderly and protect them from fraud and misinformation.

We used a previous unexplored education approach to educate the elderly - educating the younger generation and instructing them how to educate their grandparents. Compared with traditional education strategies, our approach offers emotional support from the younger generation and is comfortable for the elderly. In countries like Japan, Korea, Singapore, and China, where the bond between grandparents and grandkids is incredibly tight, our approach is undoubtedly effective.
to be replaced

To educate the youth, we have held online summer camps for high school students to pass on aged care, in addition to synthetic biology which we have been teaching in the past several years. We developed an online audio course on basic biology knowledge, which could be listened to by both the youth and the elderly.

Our free online audio course The Voice Of Biology runs on multiple popular platforms with easy access to all age groups. We incorporated graphics to attract younger audiences and provided large font textbooks to accommodate seniors and those with hearing impairment. 上传两个ogg格式的音频文件

sdg9
SDG 9: INDUSTRY, INNOVATION AND INFRASTRUCTURE

Build resilient infrastructure, promote inclusive and sustainable industrialization and foster innovation.

Our project aims at converting engineered probiotic into a sustainable calcium supplement ready for the market. Our product targets seniors suffering calcium deficiency and strives to lower the risks of osteoporosis amongst seniors. Our product is highly efficient and does not require frequent intake.

Our product is a genetically engineered probiotic that can colonize the intestine and secrete a short peptide - CaAP, which increases the amount of soluble calcium by binding to calcium ions and increasing the absorption of calcium by intestinal epithelium. Compared with other calcium supplement products such as calcium pills, our product has its unique advantages.
to be replaced

We have outlined a five-year product development plan, and signed a letter of intent with RuiShi Inc.

demo CaAP
We invented CaAP

CaAP stands for Calcium Absorption Peptide.

to be replaced
According to literature, we selected peptides GPAGPHGPVG, FDHIVY, YQEPVIAPKL, NDEELNK, DHTKE. All of these peptides can form calcium-peptide complex through their Asp, Glu and His residues, and facilitate uptake by cells. We linked these peptides through Phe-Arg (FR) junctions. When CaAP is released into the intestinal lumen, CaAP could be further cleaved into functional oligopeptides in the presence of digestive enzymes (trypsin and chymotrypsin). We contributed a basic part BBa_K3606000 to the Registry, which is RFC10 assembly compatible.

We added a signal peptide to the N terminus of CaAP to guide its secretion into the periplasmic space of E. coli. Because it is so small, it will then leak out of the cell wall into the outside environment (intestinal lumen). There are 2 existing prokaryotic signal peptides (PelB, PhoA) in the Registry, and we contributed 3 more (NSP4, OmpA, DsbA).

Driven by a quorum sensing promoter, E. coli only express signal peptide linked CaAP after their colonization. We demonstrated in vitro released CaAP by SDS-PAGE, in trichloroacetic acid precipitated bacterial culture media samples.
to be replaced

demo mcbABCD
We improved MccB17 expression

Based on previous work, we use AHL+LuxR, TetR and the antimicrobial peptide microcin B17 (MccB17) to achieve an on/off switch-like controlling of bacterial colonization and CaAP expression.

McbA,B,C,D jointly complete the secretion of MccB17. Among them, peptide product from mcbA is the precursor of the antimicrobial peptide, and McbB,C,D is responsible for the post-translational modification of McbA, to produce MccB17. McbE,F assemble the channel to export MccB17. McbG provides the immunity against MccB17. If the engineered bacteria express only McbA,B,C,D, but neither McbE,F nor McbG, intracellular accumulated MccB17 will kill itself. When the surrounding bacteria absorb MccB17 but not McbG, they would die.

We place mcbABCD under the control of a constitutive promoter, and use PtetR drive the expression of McbEFG. After the density of engineered bacteria increases to a certain extent, the heterdimers formed by the products of luxI and luxR will accumulate and activate the luxPR promoter.
to be replaced by modeling

At this time, expressed TetR transcription factor will turn off PtetR. As a result, mcbEFG stops expressing, and CaAP begins to be produced.

Improved from last year, we: (1) use low-copy plasmid backbone pFAB to reduce the expression burden on engineered bacteria; (2) low expression promoters to drive mcbABCD; (3) PtetR drives mcbEFG from the reverse strand, to avoid translational readthrough; (4) validate individual McbA/B/C/D/E/F/G expression in an IPTG induction setting.

Further, we quantitively showed the antibacterial effect with our favorite compositve part BBa_K3606029, which is RFC21 assembly compatible.
Mcb by IPTG1
In the figures above, MEFL is the absolute value of total green fluorescence and particle represents the absolute counts of bacteria, so that Y axis represents green fluorescence per bacteria. In those experiments, we mixed WT E. coli (expressing GFP driven by Plac) with mcbABCD-(mcbEFG-PtetR) at different ratios 5:7, 20:7 and 50:7, then measured MEFL/particle two hours after adding 1 mM IPTG. We used this assay (experimental flow chart at the bottom of this page) to study the antibacterial effect of MccB17 on protein synthesis.

Similarly, we shown that our antimicrobial peptide suppresses WT bacteria growth (data shown on /Team:Fudan/Parts#Composite and the main page of the part in the Registry).

flow chart of MccB17 IPTG experiments

demo CaAsst
We developed CaAsst for measuring Ca2+

Both calcium deficiency or high blood calcium leads to many diseases and severely reduces the quality of life. Using our probiotic product to cure osteoporosis might lead to calcium over supplementation. It is important to monitor blood calcium levels while using our calcium supplement.

Traditional blood calcium testing requires at least 5 mL blood and dedicated machines. We developed CaAsst (short from Calcium Assistant) to measure trace calcium ion in the blood, as little as 10 microliters. We use methylthymol blue to convert calcium concentration into 620 nm absorbance (A620). Our experimental results showed a linear relationship between calcium concentration and A620, within 20 mM calcium ion in solution, while the concentration of blood calcium ion is around 2-4 mM.
tubes with Ca2+ gradient

In addition, we introduced common interference substances to showcase the robustness of our measurement.
prism graph of Ca2+ with interference

Further development may focus on improving the stability of application solutions, making glucometer compatible test strips, and low-cost-and-portable measuring hardware.

parts
How to use our parts

Our part collection offers a complete array of parts for future researchers to develop other probiotic secreted proteins/peptides. This year, we have MccB17 and CaAP secreted and experimentally verified.

We contributed 3 probiotic signal peptides (NSP4, OmpA, DsbA), 14 low-expression promoters (BBa_K3606008 ~ BBa_K3606023) in a low-copy plasmid backbone, PtetR promoter on the reverse strand (BBa_K3606030), mcbEFG on the reverse strand (BBa_K3606821), luxPR promoter driven various proteins, PlacIq_lacI_Ptac promoter driven various proteins, PtetO3 (BBa_K3606007, improved from PtetR), etc.

We not only provide sequences and references for these parts, but also experimental results and procedures. Most of the parts were assembled together using high-fidelity PCR and ClonExpress ligation reactions. All plasmids were sequencing verified and available upon email request igem@fudan.edu.cn.

For more details, please visit:

references
Acknowledgements

Our project wouldn’t be complete without the help of many dedicated people. We were fortunate enough to have advisors, experts, stakeholders and senior undergraduate students who participated in the making of Bone appetite.

需要煽情的话

需要煽情的话

需要煽情的话

需要煽情的话

References: 只需要核心文献

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promotion video

In our team presentation video, we xxxx yyyy zzzz
team pre video