iGEM 2020 — Team Fudan

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# Team:Fudan/Notebook

[![team Fudan color logo](https://static.igem.org/mediawiki/2020/0/0e/T--Fudan--logo_2.svg)](#FudanTitleWrapper)

![notebook](https://static.igem.org/mediawiki/2020/c/c9/T--Fudan--title_notebook.svg)

Sort by Months and DNA types

* [July](#Notebook_July)
* [August](#Notebook_Augest)
* [September](#Notebook_Sep)
* [October](#Notebook_Oct)
* [DNA: Plasmids](#plasmids)
* [DNA: Primers](#primers)
* [DNA: gBlocks](#gblocks)

## Log of July

Click [here](https://static.igem.org/mediawiki/2020/b/b7/T--Fudan--Notebook_July.pdf) to download the notebook of July.

## Log of August

Click [here](https://static.igem.org/mediawiki/2020/f/f5/T--Fudan--Notebook_Augest.pdf) to download the notebook of August.

## Log of September

Click [here](https://static.igem.org/mediawiki/2020/8/81/T--Fudan--Notebook_September.pdf) to download the notebook of September.

## Log of October

Click [here](https://static.igem.org/mediawiki/2020/e/ec/T--Fudan--Notebook_October.pdf) to download the notebook of October.

## Plasmids in Stock

Click [here](https://static.igem.org/mediawiki/2020/1/1b/T--Fudan--Plasmids_Documentation.pdf) to download the PDF file. Plasmids made in October have not been frozen stock to -80 degree.

## Primers Ordered

Click [here](https://static.igem.org/mediawiki/2020/3/38/T--Fudan--Primer_Documentation.pdf) to download the PDF file.

## gBlocks Requested

```
> mcbABCD
ATGGAATTAAAAGCGAGTGaatttggtgtagttttgtccgttgatgctcttaaattatca
cgccagtctccattaggtgttggcattggtggtggtggcggcggcggcggcggcggtagc
tgcggtggtcaaggtggcggttgtggtggttgcagcaacggttgtagtggtggaaacggt
ggcagcggcggaagtggttcacatatctgatacgttgaattaaccgttcaggagcatggt
gctccctgatattaaaaaaggaaaagatatgatcaatattctgccgtttgagataatttc
cagaaatacaaagaccctgcttattacctacatttcttcagtagacattacacatgaggg
aatgaaaaaagtactcgaaagcctacgatccaaacagggtattatttctgaatacctcct
tgataaactactggacgaatcgcttattgataaagacaagggcaaagagtttcttattac
cacaggcgtgataaataaaacgaaaacatcacctctctgggtaaactcagtcataataag
cgatgttccacatcttttcagtaatgcccgggaacaatggaaatgtgatggtgtttttgt
ttctcatatcattgatataaaagataataatattaatgtgagcgattcaacactaatctg
gttacatctcgaaaactatcattcagacattgtaaaaagaatctattcaaaatttgaaag
taaccctggtgttgccttcatccaaagctactacctaaaggagtcattcaggatagatgg
agtatattcacctgatcttggtactccctgccatttttgtcatatagagagatggctgag
cagggaagaaaaaagcttcagacgtaacgaaatgtcctgggcaaacttacttcagttact
gaaaaaataccaaatgacactaccagcattggctttaggcgaatcagaaagaggattcag
ctatcatttaataaaacggcgacttcaggagttgacaggtacttcactggttaaaagtca
tgtcgataactttatgtcatctgttagcgctgatttaatcacctgtattttatgtaaaga
gccggtaattcactggcaggcttgcagctgtctggagagataacatgtcaaaacacgaac
tctctttagtggaagtaacgcattacacagatcctgaagttctggccattgttaaagatt
ttcatgtcagaggtaactttgcttccctccccgaatttgctgaacgaactttcgtgtccg
cggtacctcttgcccatctggagaaatttgaaaataaagaagttctcttcaggccaggtt
tcagctccgtaataaacatatcctcatcacataattttagtcgtgaaaggctcccatcag
gaataaacttttgcgacaaaaataaactttccattcgtactattgaaaagttattagtca
atgcattcagctcacctgatcctggctctgtaagaagaccttatccttctgggggggcat
tgtacccgattgaagtttttttatgcagattatctgaaaatacagaaaactggcaggcag
gaactaatgtttatcactacctgccgctaagtcaggcactggaacctgttgctacatgta
atactcagtcactctaccgaagcctgtccggtggggattcggaacgtcttggtaaacccc
attttgctctcgtctattgcattatttttgaaaaagctttgttcaaatatcgctacagag
gatatcggatggccttaatggaaacaggttcgatgtatcagaacgcagtattggttgcag
atcaaataggactgaaaaaccgggtatgggcgggatataccgattcatacgtagcaaaaa
caatgaatctggatcagaggactgtagcgccactgatcgttcagttttttggagatgtaa
acgatgataaatgtctacagtaaccttatgtccgcatggccggccacaatggccatgagt
ccaaaactgaacagaaatatgccaacgttttctcagatatgggactatgagcgtattaca
ccagccagcgcggccggtgaaactctgaagtcaattcagggggcaataggtgaatatttt
gaacgccgtcatttttttaatgagatagtcaccggtggtcagaaaacattatatgagatg
atgcctccatctgctgcaaaggcttttaccgaagcattttttcagatctcatcactgacc
cgcgatgaaatcataacccataaatttaaaacggtcagagcctttaatctgtttagcctt
gaacaacaagaaatacctgcagtcataattgcactcgacaatataaccgctgcagatgat
ctgaaattttatcctgacagagatacatgcggatgtagctttcatg
```

```
> mcbD-G
gtagtttgaacgatgccatagaaggttccttgtgtgaatttatggagacacagtccctcc
ttctttactggttacagggaaaagccaatactgaaatatccagtgaaatagtaacaggca
taaatcatatagatgagattttactggctctcaggtcagaaggagatatcaggattttcg
atatcaccctgcccggagctcctggacacgcagtactaaccctgtatggcacaaaaaaca
aaatcagtcgaataaaatacagtaccggattatcctatgctaatagtctgaaaaaagcac
tttgtaaatccgtagtggaattgtggcaatcgtatatatgcctgcacaactttcttattg
gcggttatactgatgatgacattattgatagttaccagcgtcactttatgtcatgcaaca
agtacgagtcgtttacggatttgtgtgaaaatacggtactactgtctgatgatgtcaagt
taacgcttgaggaaaatattacgtcagacacaaatttattaaactatcttcaacaaattt
ctgataatatttttgtttactatgccagggaaagagtaagtaacagccttgtctggtaca
caaaaatagtaagccctgattttttccttcatatgaataactcaggtgcaataaacatta
ataataaaatttaccataccggggacggtattaaagtcagagaatcaaagatggtaccat
tcccataaGGATAACAAGGTACCATTC
```

```
> FourU+relE+relB (BBa_K3036004)
ggacaagcaatgcttgccttgaatagtaacttttgaatagtgattcaggaggtactagat
gggtagcattaacctgcgtattgacgatgaacttaaagcgcgttcttacgccgcgcttga
aaaaatgggtgtaactccttctgaagcgcttcgtctcatgctcgagtatatcgctgacaa
tgaacgcttgccgttcaaacagacactcctgagtgatgaagatgctgaacttgtggagat
agtgaaagaacggcttcgtaatcctaagccagtacgtgtgacgctggatgaactccatca
tcatcatcatcactgatactagagtcacacaggaaagtactagatggcgtattttctgga
ttttgacgagcgggcactaaaggaatggcgaaagctgggctcgacggtacgtgaacagtt
gaaaaagaagctggttgaagtacttgagtcaccccggattgaagcaaacaagctccgtgg
tatgcctgattgttacaagattaagctccggtcttcaggctatcgccttgtataccaggt
tatagacgagaaagttgtcgttttcgtgatttctgttgggaaaagagaacgctcggaagt
atatagcgaggcggtcaaacgcattctccatcatcatcatcatcactaa
```

```
> NoChill06+relE+relB
cgacccttctcttctatgcgccagagacgttctagtgaaagaggagaaatactaatacta
gatgggtagcattaacctgcgtattgacgatgaacttaaagcgcgttcttacgccgcgct
tgaaaaaatgggtgtaactccttctgaagcgcttcgtctcatgctcgagtatatcgctga
caatgaacgcttgccgttcaaacagacactcctgagtgatgaagatgctgaacttgtgga
gatagtgaaagaacggcttcgtaatcctaagccagtacgtgtgacgctggatgaactcca
tcatcatcatcatcactgatactagagtcacacaggaaagtactagatggcgtattttct
ggattttgacgagcgggcactaaaggaatggcgaaagctgggctcgacggtacgtgaaca
gttgaaaaagaagctggttgaagtacttgagtcaccccggattgaagcaaacaagctccg
tggtatgcctgattgttacaagattaagctccggtcttcaggctatcgccttgtatacca
ggttatagacgagaaagttgtcgttttcgtgatttctgttgggaaaagagaacgctcgga
agtatatagcgaggcggtcaaacgcattctccatcatcatcatcatcactaa
```

```
> NoChill06+MazE+MazF
cgacccttctcttctatgcgccagagacgttctagtgaaagaggagaaatactaatacta
gatgatccacagtagcgtaaagcgttggggaaattcaccggcggtgcggattccggctac
gttaatgcaggcgctcaatctgaatattgatgatgaagtgaagattgacctggtggatgg
caaattaattattgagccagtgcgtaaagagcccgtatttacgcttgctgaactggtcaa
cgacatcacgccggaaaacctccacgagaatatcgactggggagagccgaaagataagga
agtctggtaatggtaagccgatacgtacccgatatgggcgatctgatttgggttgatttt
gacccgacaaaaggtagcgagcaagctggacatcgtccagctgttgtcctgagtcctttc
atgtacaacaacaaaacaggtatgtgtctgtgtgttccttgtacaacgcaatcaaaagga
tatccgttcgaagttgttttatccggtcaggaacgtgatggcgtagcgttagctgaccag
gtaaaaagtatcgcctggcgggcaagaggagcaacgaagaaaggaacagttgccccagag
gaattacaactcattaaagccaaaattaacgtactgattgggtagtactagagccaggca
tcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtc
ggtgaacgctctctactagagtcacactggctcaccttcgggtgggcctttctgcgttta
ta
```

Signature: [Jingqi](/Team%3AFudan/Attributions#ZhangJQ)

[![Yunfeng Capital](https://static.igem.org/mediawiki/2020/b/ba/T--Fudan--YFcapital.svg)](http://www.yfc.cn/)
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### Bone appetite: *make calcium supplement sustainable*

[Project](/Team%3AFudan/Description#Project)

* [Description](/Team%3AFudan/Description)
* [Sustainable](/Team%3AFudan/Sustainable)
* [Design](/Team%3AFudan/Design)
* [Experiments](/Team%3AFudan/Experiments)
* [Proof of concept](/Team%3AFudan/Proof_Of_Concept)

[Notebook](/Team%3AFudan/Notebook)

* [Safety](/Team%3AFudan/Safety)
* [Poster](/Team%3AFudan/Poster)
* [Promotion](https://video.igem.org/videos/watch/7a0fee51-a637-4caf-aa97-a1d9bcafd3ba)
* Presentation
* [Entrepreneurship](/Team%3AFudan/Entrepreneurship)

[Results](/Team%3AFudan/Results)

* [Contribution](/Team%3AFudan/Contribution)
* [Implementation](/Team%3AFudan/Implementation)
* [Engineering](/Team%3AFudan/Engineering)
* [Measurement](/Team%3AFudan/Measurement)
* [Modeling](/Team%3AFudan/Model)

[Parts](/Team%3AFudan/Parts)

* [Basic part](/Team%3AFudan/Parts#Basic)
* [Composite part](/Team%3AFudan/Parts#Composite)
* [Improvement](/Team%3AFudan/Improve)
* [Part collection](/Team%3AFudan/Part_Collection)

[Outreach](/Team%3AFudan/Human_Practices)

* [Integrated HP](/Team%3AFudan/Human_Practices#Integrated)
* [Education](/Team%3AFudan/Education)
* [Collaborations](/Team%3AFudan/Collaborations)
* [Inclusion](/Team%3AFudan/Inclusion)

[Team](/Team%3AFudan/Team)

* [Members](/Team%3AFudan/Team)
* [Attributions](/Team%3AFudan/Attributions)
* [Partnership](/Team%3AFudan/Partnership)
* [Acknowledgement](/Team%3AFudan/Acknowledgement)
* [Heritage](/Team%3AFudan/Heritage)

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# Team:Fudan/Engineering

[![team Fudan color logo](https://static.igem.org/mediawiki/2020/0/0e/T--Fudan--logo_2.svg)](#FudanTitleWrapper)

![engineering](https://static.igem.org/mediawiki/2020/0/06/T--Fudan--title_engineering.svg)

# Achievement in Engineering

The design of our new part CaAP (Calcium Absorption Peptide) [BBa\_K3606000](http://parts.igem.org/Part%3ABBa_K3606000) and Kill Switches ([BBa\_K3606027](http://parts.igem.org/Part%3ABBa_K3606027) and [BBa\_K3606028](http://parts.igem.org/Part%3ABBa_K3606028)) followed a engineering cycle of research and brainstorm, primitive design, debugging through discussion and consultation, further research and improved design, learn and rethink, and final design.

## Engineering of our new part CaAP (Calcium Absorption Peptide)

#### Research and Brainstorm

To solve the problem of promoting calcium absorption of intestinal epithelial cells, we did a comprehensive dig in literature at first to find out a specific factor that efficiently interacted with calcium in the digestive tract. That was when we came across calcium-binding peptides, a class of oligopeptides capable of chelating calcium, which had been found in hen egg yolk, cow milk casein, whey, soy, wheat germ and tilapia fish, *etc*. They had drawn a great deal of attention from researchers for their ability to promote calcium uptake in Caco-2 cells, indicating that they could serve as a novel kind of nutritional supplementary with high efficiency to help address the calcium deficiency problem and elevate calcium bioavailability, especially for the elderly. Moreover, the biosafety of the peptides used in our design can be guaranteed as the MTT assay have shown their absence of cytotoxicity.

However, calcium-chelating oligopeptides were quite limited while functioning independently. For instance, only a few amino acids were involved in the formation of the calcium-peptide complex. Therefore, their calcium absorption ability was not satisfying. Why not make a combination of different oligopeptides? That was where our idea of building CaAP (Calcium Absorption Peptide) originally came from.

#### Primitive design

After careful research in literature, we decided to create CaAP (Calcium Absorption Peptide) by integrating 5 different calcium-binding peptides: GPAGPHGPVG, FDHIVY, YQEPVIAPKL, NDEELNK, and DHTKE, which have been reported to enhance calcium transport into Caco-2 cell monolayers prominently as compared with the calcium alone. Among these peptides, FDHIVY, YQEPVIAPKL and NDEELNK function by forming calcium-peptide complex through Asp and Glu residues. As for DHTKE, which also facilitates the calcium influx into the cytoplasm of intestinal epithelial cells through calcium chelation, it interacts with the bivalent calcium ion by carboxyl oxygen and amino nitrogen of Asp and Glu as well as the imidazole nitrogen atoms of His residue.

#### Debugging through discussion and consultation

During the process of group discussion between our group members and consultation of teachers and advisors, we realized that there are two major problems in our primitive design.

Firstly, the one-way design of increasing calcium absorption might lead to some problems. Excessive calcium could possibly interrupt the homeostasis in our gut and bring some unwanted effects. Therefore, we decided to add a feedback circuit to the original CaAP (Calcium Absorption Peptide) design.

Secondly, we did not take the steps after CaAP production into consideration. Besides successful expression in the engineered strains (*E. coli* Nissle), the secretion of CAAP from bacteria to the intestine is of vital importance as well. Only when CAAP is eventually located in the lumen of the digestive tract can it display its robust function of combining calcium. To solve this problem, we attempted to find some signal peptides to facilitate its secretion.

#### Further research and altered design

Based on the two aforementioned problems, we directed some changes to our CaAP (Calcium Absorption Peptide) design.

As for the feedback circuit of CaAP, we decided to link a feedback factor after the CaAP sequence which is transcribed simultaneously with CaAP. The feedback factor served as a regulator of a feedback promoter. When the feedback factor gradually accumulated and reached a certain threshold, it would activate the feedback promoter and initiate the transcription of the downstream sRNA, which in turn interacted with the mRNA of CaAP and inhibit its translation. As a result, the expression of CaAP was decreased through the feedback circuit.

![engineered sRNA feedback to CaAP](https://static.igem.org/mediawiki/2020/1/10/T--Fudan--img_engineering_1.svg)

For the second problem, we decided to achieve CaAP secretion via the type II secretion system, which generally contains two steps. The first step involves some signal peptides targeted to the Sec secretory pathways, which are responsible for transporting CaAP across the inner membrane into the periplasm. For Gram-negative bacteria like *E. coli*, the second step can then take place once CaAP is in the periplasm, as the intrinsic protein complex GspD creates a pore in the outer cell membrane through which CaAP can leak out of the bacteria.

#### Learn and Rethink

When we decided to obtain a more detailed version of the feedback circuit design, we encountered some practical problems. First, it was really not an easy task for us to find a proper feedback promoter featuring a high threshold and low leakage. Otherwise, the sRNA transcription would start as soon as the CaAP and the feedback factor were transcribed, which merely reduced the upper limit of CaAP and did not execute the feedback function. Second, more importantly, the “high threshold” was actually a qualitative concept rather than a quantitative index, since the threshold was theoretically determined by the actual calcium concentration in the digestive tract, which could hardly be measured without another complex calcium-sensing system. Considering the limited laboratory time we have due to the COVID-19 pandemic, the feedback circuit is to some extent, against the engineering principle of pursuing the highest efficiency in the shortest period.

#### Final design

Eventually, we gave up the feedback circuit in the CaAP design and focused on the better expression and secretion of CaAP.

To efficiently express CaAP in the engineered strains(*E. coli*) and allow them to play a role when secreted into the extracellular space, sequences of the 5 mentioned oligopeptides are linked together through FR junctions (Phe-Arg). Hence, they could be expressed in the form of a whole peptide in the engineered bacteria and later cleaved into functional oligopeptides in the presence of digestive enzymes in the intestinal lumen. We also added 6x His tag to the C terminal of CaAP for its purification and characterization.

Meanwhile, 5 signal peptides are selected to mediate CAAP translocation through the bacterial inner membrane, including Nsp4, OmpA, DsbA, PelB, PhoA. They are all composed of an N-domain of about two to ten amino acids, a hydrophobic H-domain of about 10–20 amino acids, and a C-domain that allows for the cleavage of the signal peptidase after secretion.

Before we get our final design of CaAP, we had experienced obstacles and setbacks. Our ideas failed to prove themselves many times. However, we had been making constant attempts to use iterative engineering to step back and fix parts of our project that wasn’t working.

## Engineering of Kill Switch

#### Brainstorm

To be responsible for the environment, we considered it is necessary to introduce a Kill Switch to deprive of the survivability of engineered *E. coli* outside human bodies. Kill Switch is a gene device to eliminate bacteria in a short period of time when engineered bacteria escape from the set conditions. In the context of our project, the set condition is the human intestine and the condition under which we wanted our bacteria to die is the outside environment. Therefore, we first come up with a distinguishing condition in the intestine and in nature – glucose concentration. Glucose concentration is higher than the environment and is a very general condition that may enable the Kill Switch to apply to many situations.

#### Tentative design

After searching literature, we learned several special glucose-sensitive promoters could facilitate a different state of expression in the two conditions. We found two glucose-sensitive promoters in the part registry ([rpoH P5](http://parts.igem.org/Part%3ABBa_K3036003) and [PT-αCRP](http://parts.igem.org/Part%3ABBa_K3142012)). For example, PT-αCRP can turn on downstream genes precisely and autonomously in response to glucose limitation, and for this reason, it is also called glucose-starvation promoter. So, we imagined our Kill Switch to be a glucose-starvation promoter followed by a toxic protein gene.

#### Consultation

We further researched the parameters of the two promoters to forecast the efficiency of the Kill Switch. For one thing, the trigger concentration of them is both 0.01-0.05%, while the glucosuria level of diabetics and pregnant women can be far higher than that[1]. However, our target consumer is the elderly who also often have diabetes, glucose-starvation promoter may not be activated effectively in their excrements. For the other thing, [the model of rpoH P5](https://2019.igem.org/Team%3ABNU-China/Model) made by 19BNU-China demonstrated that glucose concentration in the intestine would drop under 0.05% within 2 hours after meals, which impeded persistent calcium supplement function[2]. For more practical information, we consulted the designer of rpoH P5 from Beijing Normal University. He didn’t consider glucose concentration as a specific signal to trigger elimination outside the body and also recommended we try toxin/antitoxin systems to enable more flexible regulation. Please visit our [Integrated Human Practices](/Team%3AFudan/Human_Practices#Integrated) for detailed information.

#### Further Research, Consultation and Redesign

After consultation, we decided to undergo a restart of our Kill Switch. Therefore, we traversed most suicide circuits in intestinal probiotics by iGEM teams, and sumarized the Response systems and the Killer systems they used. We cataloged BioBricks of the Killer system and the Response system separately and figure out the fittest part in the two groups to compose our Kill Switch. [The Kill Switch database](https://2016.igem.org/Team%3AMarburg/Modeling/Kill_Switch_Database) made by team 2016 Marburg has incredibly reduced our workload in searching Kill Switches used between 2007 and 2015. For parts of resemble mechanism, only the latest and the most efficient ones are listed. In this way, we can easily figure out the fittest part in the two groups to compose ours, and hopefully offer a reference table for the future iGEM team working on intestinal probiotics to design kill switch and choose parts more conveniently. Click [here](/Team%3AFudan/Contribution#kill_switch_tables) to see the two tables.

It consists of the toxin/antitoxin system and RNA thermometer NoChill-06 to regulate it.

![cold triggered MazF/MazE Kill Switch outside the body](https://static.igem.org/mediawiki/2020/9/95/T--Fudan--img_ks2.svg)

The antitoxin MazE is liable and expressed at a relatively high level. The MazF toxin is constitutively co-expressed with the antitoxin under the control of an RNA thermometer NoChill-06. Under the body temperature (37℃)，NoChill-06 unfolds and exposes its ribosome binding site (RBS) to express. MazE and MazF neutralize each other by protein-protein interaction and form a stable complexity in a one-to-two ratio. When the bacteria encounter a cold shock (30℃), MazE is degraded rapidly by an ATP-dependent serine protease ClpAP and releases MazF. The toxin MazF acts as a site-specific endoribonuclease to almost all cellular mRNAs, therefore resulting in cell growth arrest and finally cell death.

![model 2_5](https://static.igem.org/mediawiki/2020/f/f2/T--Fudan--model2_5.png)

Coincidentally, we found BNU-China has created a similar design in 2019. We had another consultant with BNU-China, they confirmed our design. This year, we had very limited time in laboratory, so we decided to collaborate with them, obtaining their wet lab experimental data to construct model and verify our design. We improved the logistic equation, and our model fitted the experimental data from BNU-China well. Furthermore, we adjusted the RBS properties to make an efficiency analysis. The result is that our Kill Switch obtained stronger reactions in a smaller temperature range. Click [here](/Team%3AFudan/Model) to see the modeling of our Kill Switch.

We have ordered DNA fragments from IDT in the case that we have enough time to test our Kill Switch design in wet lab. Our experimental plans to measure the growth curve of two Kill Switch were as follows:

1. Plasmids construction and transformation: Insert DNA fragments of BBa\_K3036004 and BBa\_K3606027 ordered from IDT in to pSB1C3. Transform the two kinds of constructed plasmids into DH5α strain as experimental groups and empty pSB1C plasmids as a control group. Culture three groups in 60 mL LB medium (with 50 ng/ μL ampicillin) at 37℃ overnight.
2. Cold treatment: Divide each group into two test tubes for 30℃-culture groups and 37℃-culture groups. 3 for each temperature.
3. Measure growth: Extract 5 μL bacteria solution from each test tube every 6 hours. Diluted each bacteria solution to 10^7 times and culture them on three LB plates (with 50 ng/ μL ampicillin) at 37℃ for 18 hours. Count the number of colonies in 5 cm^2 per plate after cultured for 18 hours at 37℃.
4. Draw the growth curve.

#### Learn and Improve

To find some detailed parameter to constructing a model of Kill Switch, we found literature that we didn’t notice before[3]. From this literature, we found RelE has several characteristics that need to be considered with caution. First of all, RelE is a very rare toxin that has the activity against both prokaryotic and eukaryotic organisms in a similar mechanism. It has been observed that RelE can induce apoptosis in cultured human cell lines. Besides, RelE/RelB module tends to have unexpected enrichment in bacteria that is not observed for other toxin/antitoxin modules such as MazE/MazF and ParD/ParE. Such enrichment may be associated with the formation of the persistence of microbes in the gut. In addition, the divisions of bacteria carrying homologs of the RelE toxin involved in comprising the human gut microbiota. Though all these effects only been observed in artificial mammalian gene expression systems, whether it has a tangible impact on the human gut microbiome has not been verified yet, we should be particularly careful with the safety issues.

![cold triggered MazF/MazE Kill Switch under body temperature](https://static.igem.org/mediawiki/2020/2/25/T--Fudan--img_ks4.svg)

![cold triggered MazF/MazE Kill Switch outside the body](https://static.igem.org/mediawiki/2020/a/a4/T--Fudan--img_ks3.svg)

We decided to replace the RelE/RelB system with another toxin/antitoxin system. MazF/MazE, an efficient and commonly used toxin/antitoxin module mentioned in that literature, shows no evidence of unexpected enrichment and a health threat to human. Therefore, MazF/MazE may perform better in intestinal Kill Switch. In our minds, every little improvement involving safety is a huge issue. We additionally ordered a new DNA fragment from IDT, hoping to characterize it following the protocol above in the future.

#### Reference

[1] Cowart SL, Stachura ME. Glucosuria. In: Walker HK, Hall WD, Hurst JW, editors. Clinical Methods: The History, Physical, and Laboratory Examinations. 3rd edition. Boston: Butterworths; 1990. Chapter 139. Available from: https://www.ncbi.nlm.nih.gov/books/NBK245/
[2] https://2019.igem.org/Team:BNU-China/Model#Glucose
[3] Jones, Brian V. The human gut mobile metagenome: a metazoan perspective. Gut microbes vol. 1,6 (2010): 415-31. doi:10.4161/gmic.1.6.14087

Signature: [Jingqi](/Team%3AFudan/Attributions#ZhangJQ)
[Yiyang](/Team%3AFudan/Attributions#LuYY)

[![Yunfeng Capital](https://static.igem.org/mediawiki/2020/b/ba/T--Fudan--YFcapital.svg)](http://www.yfc.cn/)
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### Bone appetite: *make calcium supplement sustainable*

[Project](/Team%3AFudan/Description#Project)

* [Description](/Team%3AFudan/Description)
* [Sustainable](/Team%3AFudan/Sustainable)
* [Design](/Team%3AFudan/Design)
* [Experiments](/Team%3AFudan/Experiments)
* [Proof of concept](/Team%3AFudan/Proof_Of_Concept)

[Notebook](/Team%3AFudan/Notebook)

* [Safety](/Team%3AFudan/Safety)
* [Poster](/Team%3AFudan/Poster)
* [Promotion](https://video.igem.org/videos/watch/7a0fee51-a637-4caf-aa97-a1d9bcafd3ba)
* Presentation
* [Entrepreneurship](/Team%3AFudan/Entrepreneurship)

[Results](/Team%3AFudan/Results)

* [Contribution](/Team%3AFudan/Contribution)
* [Implementation](/Team%3AFudan/Implementation)
* [Engineering](/Team%3AFudan/Engineering)
* [Measurement](/Team%3AFudan/Measurement)
* [Modeling](/Team%3AFudan/Model)

[Parts](/Team%3AFudan/Parts)

* [Basic part](/Team%3AFudan/Parts#Basic)
* [Composite part](/Team%3AFudan/Parts#Composite)
* [Improvement](/Team%3AFudan/Improve)
* [Part collection](/Team%3AFudan/Part_Collection)

[Outreach](/Team%3AFudan/Human_Practices)

* [Integrated HP](/Team%3AFudan/Human_Practices#Integrated)
* [Education](/Team%3AFudan/Education)
* [Collaborations](/Team%3AFudan/Collaborations)
* [Inclusion](/Team%3AFudan/Inclusion)

[Team](/Team%3AFudan/Team)

* [Members](/Team%3AFudan/Team)
* [Attributions](/Team%3AFudan/Attributions)
* [Partnership](/Team%3AFudan/Partnership)
* [Acknowledgement](/Team%3AFudan/Acknowledgement)
* [Heritage](/Team%3AFudan/Heritage)

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# Team:Fudan/Experiments

[![team Fudan color logo](https://static.igem.org/mediawiki/2020/0/0e/T--Fudan--logo_2.svg)](#FudanTitleWrapper)

![experiments](https://static.igem.org/mediawiki/2020/3/3f/T--Fudan--title_experiments.svg)

# 2020 Fudan Protocols

Click [here](https://static.igem.org/mediawiki/2020/0/0a/T--Fudan--Protocol.pdf) to download the protocols used by 2020 Fudan team, as a single PDF file.

## Cell Cryopreservation

Cryopreservation is a technique that store cells at a very low temperatures (-80℃) to reduce cell metabolic damage and enable long-term storage.

1. Add cryoprotectant: Add 80% glycerin 500 μL and bacterial fluid 1 000 μL into Cryopreservation vials. Mix upside down gently.
2. Seal the vials: Stick sellotape around the label.
3. Store the vials: Store prepared vials in refrigerator at -80℃.

## Cell Recovery

Bacteria preserved at -80℃ need to be recovery to restores cell growth.

1. Prepare plate: Take out the plate with relevant resistance from 4 degrees and wait for the temperature to the room temperature. Mark at the bottom of the plate.
2. Recover the cell: The -80℃ frozen strain was removed and placed on the ice quickly. Take a ring of the upper layer of melt bacteria liquid by the sterilized inoculation ring. Coated the plate and draw a line.
3. Culture the recovered cell: Incubate at 37℃ overnight.

## PCR

PCR is performed for amplify DNA fragments in our project.

1. Set up PCR system (25 μL):

   | Reagent | Volume |
   | --- | --- |
   | ddH2O | To 25 μL |
   | 2 x Phanta Max Buffer | 12.5 μL |
   | dNTP | 0.5 μL |
   | Phanta | 0.5 μL |
   | Template | Plasmid: 0.5 μL  PCR product: 1 μL |
   | Forward Primer | 1 μL |
   | Reverse Primer | 1 μL |
2. Place the PCR tubes in a PCR amplifier.
3. Set up reaction program:

   | Procedure | Temperature | Time | Cycle |
   | --- | --- | --- | --- |
   | Initialization | 95℃ | 30s | 1 |
   | Denaturation | 95℃ | 15s | 40 |
   | Annealing | 72℃ | 15s |
   | Extension | 72℃ | 30s/kb |
   | Final elongation | 72℃ | 5min | 1 |
4. Incubate: Incubate at 16℃ until the PCR product is picked up.

## Colony PCR

Colony PCR is performed to determine whether we insert DNA into plasmid successfully.

1. Set up colony PCR system (10 µl):

   | Reagent | Volume |
   | --- | --- |
   | ddH2O | To 10 μL |
   | 10 x Taq Buffer | 1 μL |
   | dNTP | 0.2 μL |
   | Taq DNA Polymerase | 0.2 μL |
   | Colony Template | 1 μL |
   | Forward Primer | 0.4 μL |
   | Reverse Primer | 0.4 μL |
2. Place the PCR tubes in a PCR amplifier.
3. Set up reaction program:

   | Procedure | Temperature | Time | Cycle |
   | --- | --- | --- | --- |
   | Initialization | 94℃ | 5min | 1 |
   | Denaturation | 94℃ | 30s | 25 |
   | Annealing | 72℃ | 30s |
   | Extension | 72℃ | 1min/kb |
   | Final elongation | 72℃ | 7min | 1 |
4. Incubate: Incubate at 8℃ until the PCR product is picked up.

## Overlap Extension PCR (OE PCR)

OE PCR is used to fuse fragments together.

1. First round PCR: Use primer a/b to amplify fragment AB by PCR, and c/d to amplify fragment CD. Set up PCR system:

   | Reagent | Volume |
   | --- | --- |
   | ddH2O | To 25 μL |
   | 2 x Phanta Max Buffer | 12.5 μL |
   | dNTP | 0.5 μL |
   | Phanta | 0.5 μL |
   | Template | Plasmid: 0.5 μL PCR product: 1 μL |
   | Forward Primer | 1 μL |
   | Reverse Primer | 1 μL |

   Set up reaction program:

   | Procedure | Temperature | Time | Cycle |
   | --- | --- | --- | --- |
   | Initialization | 95℃ | 30s | 1 |
   | Denaturation | 95℃ | 15s | 40 |
   | Annealing | 72℃ | 15s |
   | Extension | 72℃ | 30s/kb |
   | Final elongation | 72℃ | 5min | 1 |
2. Annealing of homologous regions in Second round PCR: Separate and purify fragment AB and CD by agarose gel electrophoresis and DNA gel extraction. Measure the concentration of the two fragments. Dilute them into 1:1 as template. Set up PCR system:

   | Reagent | Volume |
   | --- | --- |
   | ddH2O | To 50 μL |
   | 2 x Phanta Max Buffer | 25 μL |
   | dNTP | 1 μL |
   | Phanta | 1 μL |
   | Template | 0.08 pmol + 0.08 pmol |

   Set up reaction program:

   | Procedure | Temperature | Time | Cycle |
   | --- | --- | --- | --- |
   | Initialization | 94℃ | 5min | 1 |
   | Denaturation | 94℃ | 30s | 12 |
   | Annealing | 60/64/68/72℃ | 30s |
   | Extension | 72℃ | 1min/kb |
   | Final elongation | 72℃ | 7min | 1 |
3. Store the vials: Store prepared vials in refrigerator at -80℃.

## Vector PCR

Vector PCR is used for generating a linear product.

1. Set up PCR system (25 μL):

   | Reagent | Volume |
   | --- | --- |
   | ddH2O | To 25 μL |
   | 2 x Phanta Max Buffer | 12.5 μL |
   | dNTP | 0.5 μL |
   | Phanta | 0.5 μL |
   | Template | Plasmid: 0.5 μL |
   | Forward Primer | 1 μL |
   | Reverse Primer | 1 μL |
2. Place the PCR tubes in a PCR amplifier.
3. Set up reaction program:

   | Procedure | Temperature | Time | Cycle |
   | --- | --- | --- | --- |
   | Initialization | 95℃ | 30s | 1 |
   | Denaturation | 95℃ | 15s | 40 |
   | Annealing | 72℃ | 15s |
   | Extension | 72℃ | 30s/kb |
   | Final elongation | 72℃ | 5min | 1 |
4. Incubate: Incubate at 16℃ until the PCR product is picked up.

## Agarose Gel Electrophoresis

Agarose gel electrophoresis is performed to separate and confirm whether our plasmids were constructed properly.

1. Make gel solution: Add 0.7g agarose, 70ml TAE buffer into a glass bottle and heat in a micro-oven for 2 min. Cool the liquid agarose gel to lower than 60℃ and decant the liquid agarose gel into an agarose gel tank. Add 8 μL EB into the liquid agarose gel and place the electrophoresis comb.
2. Load the gel: Place the solid agarose gel in an electrophoresis device. Add 10xDNA Loading Buffer in the DNA sample, mix them up gently and carefully pipette the sample into the sample loading chambers.
3. Electrophoresis: Cover the lid of the electrophoresis device, set the electrophoresis time and start electrophoresis.

## DNA Gel Extraction

DNA Gel Extraction is to extract desired DNA from an agarose gel after agarose gel electrophoresis.

1. Cut the DNA gel: place the gel under UV light and find the DNA band of the desired nucleotide length. b) Cut the gel containing desired DNA and put it into an Eppendorf tube.
2. Melt the DNA gel: Use Vazyme® DNA Gel Extraction Kit. Add 300 μL buffer GDP to the Eppendorf tube and incubate at 55℃. Spin briefly.
3. Pure the DNA gel: Insert a Fast Mini Columns-G into a 2 ml Collection Tube, transfer the solution maximally of 700 μL once a time to a filtration column, centrifuge at 12,000 x g for 30 - 60s. Discard the filtrate and reuse the Collection Tube, add 600 μL of Buffer GW (with ethanol added) to the filtration column, centrifuge at 12,000 x g for 60s. Pure the left solution in the same way.
4. Filtrate DNA: Discard the filtrate and reuse the Collection Tube, centrifuge the empty column at 12,000 x g for 2 min. Insert the column into a clean 1.5 mL Eppendorf tube and incubate at 55℃ for 5 min. Add 7 μL - 30 μL of ddH2O (incubated at 55℃ in advance) to the center of the column membrane, incubate at room temperature for 2 min, and then centrifuge at 12,000 x g for 1min. Discard the filtration column. Measure the DNA concentration by Nanodrop 2000.
5. Store DNA at -20℃.

## Restriction Enzyme Double Digestion

Double digestion reaction is performed to create mismatch ends for directional insertion.

1. Add DNA fragment and vector at a mole ratio of 2:1 - 4:1 (total 1μg), 5 μL NEB CutSmart
   buffer, 1 μL of both restriction enzymes and ddH2O up to 50 μL to set up the reaction.
2. Incubate at 37℃ overnight.

## ClonExpress ligation reaction

ClonExpress ligation reaction is performed to insert our DNA fragments into vector.

1. Set up ClonExpress ligation system:

   | Reagent | Volume |
   | --- | --- |
   | ddH2O | To 5 μL |
   | DNA fragment | 0.02\*bp ng |
   | vector | 0.01\*bp ng |
   | 2 x ClonExpress Mix | 2.5 μL |
2. Incubate at 50℃ for 30 minutes. Transform the DNA into bacteria.

## Plasmid transformation

Plasmid transformation is performed to transfer plasmids into the host bacteria. We transform our plasmids into DH5α *E. coli* to amplify them and BL21 to testify function of our plasmids.

1. Thaw all reagents on ice. And add 20 μL competent *E. coli* cells into the ligation product.
2. Heat shock at 42℃ for 45s and then cool the mixture on ice for 2min.
3. Add 900 μL liquid SOC or LB medium (without antibiotic) into the mixture and shaking culture at 37℃ for 1h.
4. Evenly spread the liquid culture on a solid culture medium and incubate at 37℃ overnight for colonies forming on the plate.

## Plasmid Miniprep (Vazyme® FastPure Plasmid Mini Kit)

Plasmid Miniprep is performed to is extract plasmid DNA from bacteria.

1. Harvest 1 - 5 mL overnight cultured (12 - 16h) bacterial cells into a centrifuge
   tube, centrifuge at 10,000 x g for 1min, discard the supernatant and invert the tube
   on the absorbent paper to dry.
2. Add 250 μL of Buffer P1 (add RNase A before use), mix thoroughly by vortex or pipetting up and down.
3. Add 250 μL of Buffer P2, mix thoroughly by softly inverting the tube 8 - 10 times
   to assure complete lysis.
4. Add 350 μL of Buffer P3, mix gently and thoroughly by inverting the tube 8 - 10 times to neutralize Buffer P2 until a flocculent white precipitate forms and centrifuge at 13,000 x g for 10min.
5. Insert a FastPure DNA Mini Column into a 2 mL Collection Tube, transfer the supernatant from step 4 to the Filtration Column, centrifuge at 13,000 x g for 30 - 60s, discard the filtrate and reuse the Collection Tube.
6. Add 600 μL of Buffer PW2 (with ethanol added in) to the Filtration Column, centrifuge at 13,000 x g for 30 - 60s, discard the filtrate and reuse the Collection Tube. Centrifuge the empty Filtration Column for l min at 13,000 x g.
7. Insert the Column into a clean 1.5 mL microcentrifuge tube, add 30 - 100 μL of Elution Buffer to the center of the Column membrane, incubate at room temperature for 2min, centrifuge at 13,000 x g for 1min. Discard the Filtration Columns, store DNA at -20℃.

## SDS-PAGE

SDS-PAGE is performed for the separation of polypeptides and confirm whether our circuits expressed properly.

1. Prepare 10ml 10% Running Gel solution: Add4.1 mL ddH2O, 3.3 mL 30% Acrylamide/Bis (29:1 or 37.5:1), 2.5 mL 1.5 M Tris-HCl pH8.8, 100 μL 10% SDS, 50 μL 10% APS, 5 μL TEMED. Mix them thoroughly.
2. Prepare 2ml 4% Stacking Gel solution: Add 1.22 ml ddH2O, 0.26ml 30% Acrylamide/Bis (29:1 or 37.5:1), 0.5 mL 0.5 M Tris-HCl pH6.8, 20 μL 10% SDS, 20 μL 10% APS, 2 μL TEMED. Mix them thoroughly.
3. Prepare the protein sample: Add 1 ml bacterial cells per time into a centrifuge tube, centrifuge at 12,000 x g for 1min, discard the supernatant. Dilute SDS sample buffer to 1x. Add 1 x SDS Loading to 3000 cells per 1 μL, mix thoroughly by vortex or pipetting up and down until there is no visible precipitation. Incubate at 99℃ for 5min.
4. Make the gel: Assemble the gel cassette and make sure it not to leak. Fill the gel cassette with the Running Gel softly and fill up the cassette with ddH2O. Keep it still for 10 - 20min until the water layer can be observed. Pour out the ddH2O completely and fill up the cassette with Stacking Gel. Insert the comb and take care not to catch bubbles under the teeth. Keep it still.
5. Load the gel: Take off the cassette and assemble the gel running stand. Fill the stand with 1 x SDS running buffer and remove the combs from the gel. Mix up Marker with 1 x SDS Loading. Load 15 μL marker mixture into the wells.
6. Electrophoresis: Cover the lid of the electrophoresis device, and start electrophoresis at 200 V until the dye front is nearly at the bottom of the gel.
7. Stain the gel: Submerge the whole piece of the disassembled gel with 0.1% Coomassie Blue dye for 30min.
8. Destain the gel: Pour out the 0.1% Coomassie Blue dye and wash it using ddH2O. Destain with destaining solution for 30min. Change the destaining solution to destain until it clear.
9. Scan the gel.

## SDS-PAGE Gradient Gel

SDS-PAGE is performed for the separation of caap and confirm whether our circuits functioned properly.

1. Start the device: Put the container on the electromagnetic heating device. Increase the speed of the rotor gradually to 260 RPM.
2. Prepare Gel solution: 4% and 20% Gel solution, can be prepared directly according to SDS-PAGE protocol. And 1.5 times of APS concentration should be added.
3. Gradient formation: Add 4% glue slowly into the valve, and then add 20% glue. Mix them by the rotor. Wait about 2 hours for the glue to concrete.
4. Store the gel: Remove the solidified glue one by one. Soak them in 1×SDS, and put them into a refrigerator at 4℃ under the counter for preservation.

## IPTG induction experiment

1. Reconnection: Take 10 mL and 5 mL LB liquid medium in two test tubes, 10 mL one is used as control group and 5 mL one is for experiment group. Add 500× ampicillin 20 μL and 10 μL, respectively. Culture 6h. Add 1 mL bacteria solution to experiment group and divided evenly into two tubes (3 mL each) for ipTG-induced and non-induced groups. Another 1 mL bacteria was used to measure initial OD.
2. Measure OD: Measured OD value once per 30min and once per 15min after OD reached 0.2.
3. Induce: When OD value reached about 0.6, add 500mM IPTG reach final concentration of 1 mM. Incubate for 3 hours.

## Anhydrotetracycline induction experiment

1. Dilute aTc: Dilute 2 mg/ mL aTc solution into 100 ng/ mL, 200 ng/ mL and 400 ng/ mL.
2. Induce: Add 3 mL LB medium (with kan), 200 μL ON culture and 32 μL corresponding inducer (in one of the test tubes add 32 μL LB medium as control) into 3 mL bacteria solution.
3. Measure: Add 300 μL of each test tube to the microplate reader in triplet, and measure the t0 at the correct wavelength and OD600. Put the test tubes in the shaker at 37℃ and 250 rpm. After 4 hours measure the t4, after 8 hours measure the t8 and after 12h measure the t12.

## Growth Curve Measurement

Growth Curve Measurement is used to characterize our Kill Switches.

1. Plasmids construction and transformation: Insert DNA fragments of BBa\_K3036004, BBa\_K3606027 and BBa\_K3606028 into pSB1C3. Transform the two kinds of constructed plasmids into DH5α strain as experimental groups and empty pSB1C plasmids as control group. Culture three groups in 60mL LB medium (with 50 ng/ μL ampicillin) at 37℃ overnight.
2. Cold treatment: Divide each group into two test tubes for 30℃-culture groups and 37℃-culture groups. (3 for each temperature).
3. Measure growth situation: Extract 5 μL bacteria solution from each test tube every 1h. Diluted each bacteria solution to 10^7 times and culture them on three LB plate (with 50 ng/ μL ampicillin) at 37℃ for 24h. Count the number of colonies in 5 cm^2 per plate after cultured for 24h at 37℃.
4. Draw the growth curve.

## Methylthymol Blue Method for Blood Calcium Measurement

Methylthymol Blue Method is used to determine the concentration of calcium chromogenic reaction.

1. Prepare solution 1

   | Reagent | Volume |
   | --- | --- |
   | H2O | To 45 mL |
   | MTB | 0.009 g |
   | 8-Hydroxyquinoline | 6.096 g |
   | HCl | 0.55785 mL |
   | Urea | 8.109g |
2. Prepare solution 2

| Reagent | Volume |
| --- | --- |
| H2O | To 45 mL |
| Ethanolamine | 9 mL |
| Urea | 8.109g |

3. Set up chromogenic reaction: Take 1ml solution 1 and 1ml solution 2 to make the application solution. Add 34 μL standard calcium ion sample into the system in proportion of 60:1 and mix it.
4. Measure the absorbance: Wait for about 3 to 5 min and measure the absorbance of the solution with a spectrophotometer.
5. Change the standard calcium ion concentration from 0 to 20 mM and make multiple measurements to obtain the linearity curve.

Click [here](https://static.igem.org/mediawiki/2020/0/0a/T--Fudan--Protocol.pdf) to download the protocols used by 2020 Fudan team, as a single PDF file.

Signature: [Jingqi](/Team%3AFudan/Attributions#ZhangJQ)

[![Yunfeng Capital](https://static.igem.org/mediawiki/2020/b/ba/T--Fudan--YFcapital.svg)](http://www.yfc.cn/)
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[![School of Life Sciences, Fudan University](https://static.igem.org/mediawiki/2018/1/1d/T--Fudan--schoolOfLifeSciencesIcon.png)](http://life.fudan.edu.cn/)

### Bone appetite: *make calcium supplement sustainable*

[Project](/Team%3AFudan/Description#Project)

* [Description](/Team%3AFudan/Description)
* [Sustainable](/Team%3AFudan/Sustainable)
* [Design](/Team%3AFudan/Design)
* [Experiments](/Team%3AFudan/Experiments)
* [Proof of concept](/Team%3AFudan/Proof_Of_Concept)

[Notebook](/Team%3AFudan/Notebook)

* [Safety](/Team%3AFudan/Safety)
* [Poster](/Team%3AFudan/Poster)
* [Promotion](https://video.igem.org/videos/watch/7a0fee51-a637-4caf-aa97-a1d9bcafd3ba)
* Presentation
* [Entrepreneurship](/Team%3AFudan/Entrepreneurship)

[Results](/Team%3AFudan/Results)

* [Contribution](/Team%3AFudan/Contribution)
* [Implementation](/Team%3AFudan/Implementation)
* [Engineering](/Team%3AFudan/Engineering)
* [Measurement](/Team%3AFudan/Measurement)
* [Modeling](/Team%3AFudan/Model)

[Parts](/Team%3AFudan/Parts)

* [Basic part](/Team%3AFudan/Parts#Basic)
* [Composite part](/Team%3AFudan/Parts#Composite)
* [Improvement](/Team%3AFudan/Improve)
* [Part collection](/Team%3AFudan/Part_Collection)

[Outreach](/Team%3AFudan/Human_Practices)

* [Integrated HP](/Team%3AFudan/Human_Practices#Integrated)
* [Education](/Team%3AFudan/Education)
* [Collaborations](/Team%3AFudan/Collaborations)
* [Inclusion](/Team%3AFudan/Inclusion)

[Team](/Team%3AFudan/Team)

* [Members](/Team%3AFudan/Team)
* [Attributions](/Team%3AFudan/Attributions)
* [Partnership](/Team%3AFudan/Partnership)
* [Acknowledgement](/Team%3AFudan/Acknowledgement)
* [Heritage](/Team%3AFudan/Heritage)

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# Team:Fudan/Results

[![team Fudan color logo](https://static.igem.org/mediawiki/2020/0/0e/T--Fudan--logo_2.svg)](#FudanTitleWrapper)

![results](https://static.igem.org/mediawiki/2020/5/50/T--Fudan--title_results.svg)

## Improvement of the antimicrobial peptide expressing system

Here, we tried to improve the former antimicrobial peptide (Mccb17) expression, based on 2019 Fudan [BBa\_K3245010](http://parts.igem.org/Part%3ABBa_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](/Team%3AFudan/Design) 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](https://static.igem.org/mediawiki/2020/d/d5/T--Fudan--img_flowchart1.svg)

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](https://static.igem.org/mediawiki/2020/1/16/T--Fudan--5mix7.jpeg)

###### Figure 2. MEFL/particle for different Px and PxAD with the ratio of mixure WT:Px=5:7

![results IPTG 20 7](https://static.igem.org/mediawiki/2020/b/b6/T--Fudan--20mix7.jpeg)

###### Figure 3. MEFL/particle for different Px and PxAD with the ratio of mixure WT:Px=20:7

![results IPTG 50 7](https://static.igem.org/mediawiki/2020/8/89/T--Fudan--50mix7.jpeg)

###### 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](https://static.igem.org/mediawiki/2020/6/61/T--Fudan--flowchart_kanamp.svg)

###### 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](https://static.igem.org/mediawiki/2020/b/b9/T--Fudan--AUP21.jpeg)

###### 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](http://parts.igem.org/Part%3ABBa_K3606006)), PtetO3 ([BBa\_K3606007](http://parts.igem.org/Part%3ABBa_K3606007)) along with the original PtetR ([BBa\_R0040](http://parts.igem.org/Part%3ABBa_R0040)) via plate reader by combining them with GFP, when they were induced by different concentration of anhydrotetracycline (aTc) or not induced.

![figure 1](https://static.igem.org/mediawiki/2020/5/51/T--Fudan--aTc_0.jpg)

###### 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](https://static.igem.org/mediawiki/2020/d/d3/T--Fudan--aTc_200.jpg)

###### 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](https://static.igem.org/mediawiki/2020/0/06/T--Fudan--aTc_100.jpg)

###### 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](https://static.igem.org/mediawiki/2020/4/46/T--Fudan--aTc_400.jpg)

###### 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](https://static.igem.org/mediawiki/parts/7/7a/T--Fudan--rate_100.jpg)

###### Figure 11. When aTc=100, fold of increase of promoters

![figure 6](https://static.igem.org/mediawiki/2020/a/a4/T--Fudan--rate_200.jpg)

###### Figure 12. When aTc=200, fold of increase of promoters

![figure 7](https://static.igem.org/mediawiki/2020/0/0f/T--Fudan--rate_400.jpg)

###### 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](/Team%3AFudan/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](http://parts.igem.org/Part%3ABBa_K3606042) |
| OmpA | [BBa\_K3606043](http://parts.igem.org/Part%3ABBa_K3606043) |
| DsbA | [BBa\_K3606030](http://parts.igem.org/Part%3ABBa_K3606030) |
| PelB | [BBa\_K208004](http://parts.igem.org/Part%3ABBa_K208004) |
| PhoA | [BBa\_K808028](http://parts.igem.org/Part%3ABBa_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](https://static.igem.org/mediawiki/2020/0/05/T--Fudan--denoted_gel2_OmpA_MBP.png)

![1_1](https://static.igem.org/mediawiki/2020/3/3b/T--Fudan--denoted_gel3_OmpA_MBP.png)

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](https://static.igem.org/mediawiki/parts/6/67/T--Fudan--denoted_gel2_signalpeptides.png)

###### Figure 16. NSP4/PhoA/OmpA-CaAP expression with and without IPTG induction in the bacterial lysate.

![2_6](https://static.igem.org/mediawiki/parts/0/0d/T--Fudan--File-T--Fudan--denoted_gel3_signalpeptides.png)

###### 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](/Team%3AFudan/Model), and our online survey results are at [/Team:Fudan/Human\_Practices](/Team%3AFudan/Human_Practices).

Signature:
[Zhenru](/Team%3AFudan/Attributions#ZhangZR)
[Gaochen](/Team%3AFudan/Attributions#ZhangGC)

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### Bone appetite: *make calcium supplement sustainable*

[Project](/Team%3AFudan/Description#Project)

* [Description](/Team%3AFudan/Description)
* [Sustainable](/Team%3AFudan/Sustainable)
* [Design](/Team%3AFudan/Design)
* [Experiments](/Team%3AFudan/Experiments)
* [Proof of concept](/Team%3AFudan/Proof_Of_Concept)

[Notebook](/Team%3AFudan/Notebook)

* [Safety](/Team%3AFudan/Safety)
* [Poster](/Team%3AFudan/Poster)
* [Promotion](https://video.igem.org/videos/watch/7a0fee51-a637-4caf-aa97-a1d9bcafd3ba)
* Presentation
* [Entrepreneurship](/Team%3AFudan/Entrepreneurship)

[Results](/Team%3AFudan/Results)

* [Contribution](/Team%3AFudan/Contribution)
* [Implementation](/Team%3AFudan/Implementation)
* [Engineering](/Team%3AFudan/Engineering)
* [Measurement](/Team%3AFudan/Measurement)
* [Modeling](/Team%3AFudan/Model)

[Parts](/Team%3AFudan/Parts)

* [Basic part](/Team%3AFudan/Parts#Basic)
* [Composite part](/Team%3AFudan/Parts#Composite)
* [Improvement](/Team%3AFudan/Improve)
* [Part collection](/Team%3AFudan/Part_Collection)

[Outreach](/Team%3AFudan/Human_Practices)

* [Integrated HP](/Team%3AFudan/Human_Practices#Integrated)
* [Education](/Team%3AFudan/Education)
* [Collaborations](/Team%3AFudan/Collaborations)
* [Inclusion](/Team%3AFudan/Inclusion)

[Team](/Team%3AFudan/Team)

* [Members](/Team%3AFudan/Team)
* [Attributions](/Team%3AFudan/Attributions)
* [Partnership](/Team%3AFudan/Partnership)
* [Acknowledgement](/Team%3AFudan/Acknowledgement)
* [Heritage](/Team%3AFudan/Heritage)

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