iGEM 2021 — Team Fudan

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# Team:Fudan/Notebook

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# Notebook

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* Team
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* Lab
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---

## Done by the Team

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# Team:Fudan/Engineering

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# Engineering

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# [#](#engineering-success-for-inhibiting-protein-expression-in-e-coli) Inhibiting protein expression in *E. coli*

## [#](#overview) Overview

In order to achieve the goal of high levels of expression of any target protein in the chassis organism and thus obtain the highest concentration (best purify without protein purification procedure) of the target protein,
we need to globally inhibit all pbackground rotein expression except the gene of interest in *E. coli*.

## [#](#engineering-cycle) Engineering Cycle

## [#](#design) Design

Phages are known to be capable of reducing the expression of the chassis biological background proteins. We reviewed the literature and found that the T7 phage could achieve this through three proteins, gene protein (gp)
0.7, gp2 and gp5.7 . Gp0.7 and gp2 are proteins that function early during phage infection, while gp5.7 is a highly active protein in the middle of the phage infection. Phages can suppress the expression of native proteins
early in the infection, and thus we believe that gp5.7 plays a complementary function to the gp2 protein, and that gp2 may has a stronger effect of reducing the background protein expression level. Therefore, we initially
entailed gp2 in our circuits. As for gp0.7, it is reported as a broad-spectrum protein kinase and therefore can inhibit RNAP while interfering with the function of many other proteins, and we were aleither not sure
if it would affect the target protein (Bst in our case). Besides, Gp0.7 has a large molecular weight. The host bacterium is under more stress when expressing it, so we did not select gp0.7. In contrast, gp2 is a relatively
small molecular weight protein, with only 198 amino acids. For the above reasons, we chose gp2 to act as the inhibitor of background protein in our engineering bacteria.

However, leakage is also a severe problem that we predicted to encounter. To prevent the leakage of gp2 protein and inhibiting the growth of host bacteria before induction, we chose a medium copy of replication start and
p15A Ori[1] and a low leakage promoter pBAD [2]. in our circuits.

## [#](#build) Build

We synthesized gp2 directly with Oligo Assembly for it is a small protein, P15A ori from the plysS plasmid, and pBAD sourced from the Distribution Kit our team received in 2019. we then used Overlab PCR and homologous recombination
to ligate these fragments together and obtain a complete loop capable of fulfilling the intended function of the plasmid. In addition, to characterize the amount of heteroprotein, we obtained a plasmid with pCVD as
a vector attached to a σ70 constitutive promoter controlling GFP expression. These two plasmids were cotransfected with Fast T1 *E. coli* (a modified version of DH5α) to obtain *E. coli* for culture.

![pic](https://static.igem.org/mediawiki/2021/b/b9/T--Fudan--engsup1.png)
Figure 1. araC-gp2-plysS

![pic](https://static.igem.org/mediawiki/2021/2/27/T--Fudan--engsup2.png)
Figure 2. GFP-pCVD

## [#](#test) Test

We obtained *E. coli* successfully transformed with a functional plasmid and then cultured. We added 0, 10 mM, 1 mM, 0.1 mM, 0.01 mM arabinose at the absorbance OD600 of 0.2, and continued to incubate the bacteria,
and measured the OD600 and GFP fluorescence intensity (excitation wavelength 488 nm, emission wavelength 530 nm) at half-hour intervals for 12 h. The results showed that the growth status and the expression of GFP in
the bacterium under different level of induction were quite similar, indicating that the background protein has strongly inhibited the transcription of *E. coli* without induction,.We speculated that our anti-leakage
measures failed to completely prevent the leakage of gp2.

![https://static.igem.org/mediawiki/parts/0/0d/T--Fudan--K3790231fig1.png](https://static.igem.org/mediawiki/parts/0/0d/T--Fudan--K3790231fig1.png)

Figure 3. Growth curve under different induction on gp2 expression (Mean±SD)

![https://static.igem.org/mediawiki/parts/1/1c/T--Fudan--K3790231fig2.png](https://static.igem.org/mediawiki/parts/1/1c/T--Fudan--K3790231fig2.png)

Figure 4. σ70 driven GFP expression under different level of arabinose induction on gp2 expression (Mean±SD)

## [#](#learn) Learn

We learned that the function of gp2 was too strong that even minor leakage will severely affect the growth of our engineering bacteria.

To solve the problem that the repressor protein strongly inhibits *E. coli* transcription even without induction, due to its leakage expression, we can on the one hand try to optimize the anti-leakage element, and on the
other hand, reduce the function of the repressor protein so that it won't cause notable effects when not induced even there's some leakage.

## [#](#design-2) Design

We think it's more practical to reduce the function of the repressor protein being expressed. To realize this, we chose gp5.7 to substitute for gp2, because gp5.7 is mainly a σS inhibitor, which can only slightly inhibit
σ70.Even if the leakage of σS cause uncontrollable inhibition to transcription during growth plateau, it doesn't matter since the cell density has reached the up-limit. So, this design can well solve the problem of
over-functioning transcriptional repressor protein.

## [#](#build-2) Build

We replaced the gp2 protein with gp5.7 protein and transfected *E. coli* along with other components.

![pic](https://static.igem.org/mediawiki/2021/9/99/T--Fudan--engfig3gp5.7.png)
Figure 5. araC-gp5.7-plysS

## [#](#test-2) Test

We obtained transfected *E. coli* after incubation under the same conditions and measured OD600 and GFP fluorescence intensity after arabinose induction. We found significant differences in the growth curve and GFP
expression (no matter it's driven by σ70 or σS) of the organism without induction and with different concentrations of induction. It proved that we reached the goal that the expression of the native protein could be
inhibited only by induction at specific concentrations.

![https://static.igem.org/mediawiki/parts/thumb/c/ca/T--Fudan--K3790232fig4.png/900px-T--Fudan--K3790232fig4.png](https://static.igem.org/mediawiki/parts/thumb/c/ca/T--Fudan--K3790232fig4.png/900px-T--Fudan--K3790232fig4.png)

Figure 6. Growth curve under different induction on gp5.7 expression (Mean±SD)

![https://static.igem.org/mediawiki/parts/thumb/8/85/T--Fudan--K3790232fig5.png/900px-T--Fudan--K3790232fig5.png](https://static.igem.org/mediawiki/parts/thumb/8/85/T--Fudan--K3790232fig5.png/900px-T--Fudan--K3790232fig5.png)

Figure 7. σ70 driven GFP expression under different level of arabinose induction on gp5.7 expression (Mean±SD)

![https://static.igem.org/mediawiki/parts/thumb/0/04/T--Fudan--K3790014fig8.png/900px-T--Fudan--K3790014fig8.png](https://static.igem.org/mediawiki/parts/thumb/0/04/T--Fudan--K3790014fig8.png/900px-T--Fudan--K3790014fig8.png)

Figure 8. σS driven GFP expression under different level of arabinose induction on gp5.7 expression (Mean±SD)

## [#](#learn-2) Learn

Although the initial goal has been achieved, we can still try other anti-leakage methods to more precisely regulate the expression of gp5.7 protein.

# [#](#engineering-success-for-detecting-the-infection-of-C. albicans) Detecting the infection of *C. albicans*

## [#](#overview-2) Overview

In order to specifically detect *Candida albicans,* we used isothermal amplification to amplify their specific fragments for lateral flow assay.

## [#](#engineering-cycle-2) Engineering Cycle

## [#](#design-3) Design

Because of the small number of samples tested, we had to introduce an easily implemented isothermal amplification reaction to amplify the sample. Amplification reaction volume, only need to meet the minimum lower limit
of the lateral flow assay test strips. Initially, we reasoned the primers for loop-mediated isothermal amplification (LAMP) were too complex to design and chose thermophilic helicase-dependent isothermal amplification
(tHDA) for isothermal amplification to detect *Candida albicans* 5.8s rDNA.

## [#](#build-3) Build

tHDA primer1: 5-CTGGGTTTGGTGTTGAGCAATACGACT

tHDA primer2: 5-CCGCAAGCAATGTTTTTGGTTAGACCT

We purchased tHDA enzyme from NEB (#H0110S).

## [#](#test-3) Test

However, after more than two months of shooting and seeking help from various parties, including support from NEB, we still could not solve the problem that the signal of the amplification bands of the commercial tHDA kit
was extremely weak and difficult to detect.

![pic](https://static.igem.org/mediawiki/2021/f/f1/T--Fudan--engfig5.png)
Figure 9. Results of commercial tHDA of *Candida albicans* 5.8s rDNA.

## [#](#learn-3) Learn

When we contacted customer service, we were told that the tHDA amplification bands were just very weak and our experiments found it difficult to use the tHDA amplification products for subsequent LFA experiments, so we
switched to other isothermal amplification methods.

![pic](https://static.igem.org/mediawiki/2021/e/e6/T--Fudan--engfig6.png)
Figure 10. Screenshot of the email from NEB customer service answering questions

We interviewed Prof. [Yongming Wang](https://life.fudan.edu.cn/e7/3d/c28175a386877/page.htm), whose research interest includes combining LAMP with CRISPR Cas9 to carry out detections.

According to Prof. Wang’s insight, LAMP and RPA are potentially better alternatives to our tHDA method. When testing RNA viruses, rt-RPA was shown to yield false-positive results, while LAMP is relatively more sensitive
and cost-efficient.

## [#](#design-4) Design

We noticed that tHDA requires two types of enzymes, decarboxylase, and polymerase, both of which are temperature sensitive - there is no reliable route to transport to rural regions. We then want to produce and improve
enzymes using synthetic biology methods, we turned our attention to LAMP, the simplest enzyme, which amplifies much more efficiently than tHDA, and although its primers are more complex, it also has better reaction
specificity, recognizing six nucleic acid fragments specifically.

![pic](https://static.igem.org/mediawiki/2021/b/be/T--Fudan--engfig7.png)
Table 1. Comparison of LAMP and tHDA[3]

## [#](#build-4) Build

After bioinformatics analysis of the genome DNA using BLAST and referencing the existing PCR detection method of *C. albicans*, we targeted the ITS2 region of the 5.8S rDNA of *C.* *albicans*, and the
primers and probe are as follow:

|  | Sequenc (5--3) | Type |
| --- | --- | --- |
| F3 | TGGGTTTGCTTGAAAGACGG | LAMP Primer |
| B3 | CTTCACTCGCCGCTACTG |  |
| FIP | CCGCCGCAAGCAATGTTTTTGGTAGTGGTAAGGCGGGATCG |  |
| biotin-BIP | ATCAGGTAGGACTACCCGCTGAAGGCAATCCCTGTTGGTTTC |  |
| LF | TTGACAATGGCTTAGGTCTA |  |
| LB | GCATATCAATAAGCGGAGGA |  |
| FITC-probe | ATTGCTTGCGGCGGTAACGTCC | LFA Probe |

The LAMP primer sets were designed using Primer Explorer Ver. 4 (http://primerexplorer.jp/e/). For lateral flow assay (LF) Assay detection, one
detection probe was tagged with biotin at the 5' end, which has been entailed in the primer, and the other with FITC at the 3' end. The newly designed probes were screened using the oligoanalyzer and BLAST tool (
http://www.ncbi.nlm.nih.gov/BLAST).

## [#](#test-4) Test

Eventually, we switched to LAMP for the isothermal amplification reaction and obtained positive results with better specificity and stronger signal.

![pic](https://static.igem.org/mediawiki/2021/9/9a/T--Fudan--engfig8.png)
Figure 11. A successful test of LAMP combined with LFA. M: DL2000 marker, The brightest band of 750 bp was about 100 ng and other
bands about 50 ng. N: negative control, using genomic DNA of *S. cerevisiae* as the sample); C: positive amplification of the genomic DNA of *C. alibicans*, which displayed a red line at the appropriate
position on LFA testing strip.

# [#](#reference) Reference

[1]. Sathiamoorthy S, Shin JA. Boundaries of the origin of replication: creation of a pET-28a-derived vector with p15A copy control allowing compatible coexistence with pET vectors. *PLoS One*. 2012;7(10):e47259.
doi:10.1371/journal.pone.0047259

[2]. Guzman LM, Belin D, Carson MJ, Beckwith J. Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. *J Bacteriol*. 1995;177(14):4121-4130. doi:10.1128/jb.177.14.4121-4130.1995

[3]. Zhao, Y., Chen, F., Li, Q., Wang, L., & Fan, C. (2015). Isothermal Amplification of Nucleic Acids. *Chemical reviews*, *115*(22), 12491–12545.

1. [1. Inhibiting protein expression in *E. coli*](#engineering-success-for-inhibiting-protein-expression-in-e-coli)
   1. [1.1 Overview](#overview)
   2. [1.2 Engineering Cycle](#engineering-cycle)
   3. [1.3 Design](#design)
   4. [1.4 Build](#build)
   5. [1.5 Test](#test)
   6. [1.6 Learn](#learn)
   7. [1.7 Design](#design-2)
   8. [1.8 Build](#build-2)
   9. [1.9 Test](#test-2)
   10. [1.10 Learn](#learn-2)
2. [2. Detecting the infection of *C. albicans*](#engineering-success-for-detecting-the-infection-of-C. albicans)
   1. [2.1 Overview](#overview-2)
   2. [2.2 Engineering Cycle](#engineering-cycle-2)
   3. [2.3 Design](#design-3)
   4. [2.4 Build](#build-3)
   5. [2.5 Test](#test-3)
   6. [2.6 Learn](#learn-3)
   7. [2.7 Design](#design-4)
   8. [2.8 Build](#build-4)
   9. [2.9 Test](#test-4)
3. [3. Reference](#reference)

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* Team
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* Lab
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* Human Practices
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  + [Sustainable](/Team%3AFudan/Sustainable)

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---

## Written by: Guonan Cai

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# Team:Fudan/Experiments

![](https://static.igem.org/mediawiki/2021/a/a6/T--Fudan--Star.png)
![](https://static.igem.org/mediawiki/2021/6/65/T--Fudan--Candida.png)

# Experiments

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# [#](#2021-fudan-protocols) 2021 Fudan Protocols

Click to download the protocols used by the 2020 Fudan team, as a single PDF file.

## [#](#cell-cryopreservation) Cell Cryopreservation

Cryopreservation is a technique that stores cells at a very low temperature (-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 the refrigerator at -80℃.

## [#](#cell-recovery) Cell Recovery

Bacteria preserved at -80℃ need to be recovered 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

PCR is performed to 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 DNA polymerase | 0.5 μL |
| DNA Template | <br/>Plasmid: 0.5 μL<br/><br/>PCR product: 1 μL<br/> |
| 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 | 30-40 |
| Annealing | depends on the primers | 15s |  |
| Extension | 72℃ | 30-60s/kb |  |
| Final elongation | 72℃ | 5min | 1 |

4. Incubate: Incubate at 16℃ until the PCR product is picked up.

## [#](#colony-pcr) Colony PCR

Colony PCR is performed to determine whether we insert DNA into the 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 and set up a reaction program:

| Procedure | Temperature | Time | Cycle |
| --- | --- | --- | --- |
| Initialization | 94℃ | 5min | 1 |
| Denaturation | 94℃ | 30s | 25 |
| Annealing | depends on the primers | 30s |  |
| Extension | 72℃ | 1min/kb |  |
| Final elongation | 72℃ | 7min | 1 |

3. Incubate: Incubate at 8℃ until the PCR product is picked up.

## [#](#overlap-extension-pcr-oe-pcr) Overlap Extension PCR (OE PCR)

OE PCR is used to fuse fragments.

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 DNA polymerase | 0.5 μL |
| DNA Template | Plasmid: 0.5 μL<br/>PCR product: 1 μL |
| Forward Primer | 1 μL |
| Reverse Primer | 1 μL |

2. Set up reaction program:

| Procedure | Temperature | Time | Cycle |
| --- | --- | --- | --- |
| Initialization | 95℃ | 30s | 1 |
| Denaturation | 95℃ | 15s | 30-40 |
| Annealing | depends on the primers | 15s |  |
| Extension | 72℃ | 30s/kb |  |
| Final elongation | 72℃ | 5min | 1 |

3. 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 a 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 |

4. 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 |

5. Store the vials: Store prepared vials in the refrigerator at -80℃.

## [#](#vector-pcr) 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 | depends on the primers | 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

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

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) 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

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

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 shake 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 (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

SDS-PAGE is performed for the separation of polypeptides and confirms 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 does not 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 a destaining solution for 30min. Change the destaining solution to destain until it is clear.
9. Scan the gel.

## [#](#iptg-induction-experiment) IPTG induction experiment

1. Reconnection: Take 10 mL and 5 mL LB liquid medium in two test tubes, 10 mL one is used as the control group and 5 mL one is for the experiment group. Add 500× ampicillin 20 μL and 10 μL, respectively. Culture 6h. Add
   1 mL bacteria solution to the experiment group and divide 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 to reach the final concentration of 1 mM. Incubate for 3 hours.

## [#](#growth-curve-measurement) Growth Curve Measurement

Growth Curve Measurement is used to identify the type of promoter reported in a paper to determine whether it is a σs or σ70 promoter.

1. Plasmids transformation and cultivation: Transform the constructed plasmids into DH5α strain. Culture three groups in 60mL LB medium (with 50 ng/ μL ampicillin) at 37℃ overnight.
2. Dilute the bacteria solution to 1% with 100ml LB culture medium, and shake for certain hours as needed.
3. Obtain 300ul of sample every 30 minutes, and store it in 4℃.
4. Measure the OD600 of each sample using a plate reader. Draw the growth curve.

## [#](#fluorescence-intensity-curve-measurement) Fluorescence Intensity Curve Measurement

1. Plasmids transformation and cultivation: Transform the constructed plasmids into DH5α strain. Culture three groups in 60mL LB medium (with 50 ng/ μL ampicillin) at 37℃ overnight.
2. Dilute the bacteria solution to 1% with 100ml LB culture medium, and shake for certain hours as needed.
3. Obtain 300ul of sample every 30 minutes, and store it in 4℃.
4. Measure the fluorescence intensity of each sample using a plate reader. Draw the growth curve.

## [#](#loop-mediated-isothermal-amplification-lamp) Loop-mediated isothermal amplification (LAMP)

1. Prepare LAMP reaction system as below

| 10x Bst LF Buffer | 2.5ul |
| --- | --- |
| F3/B3/LF/LB | 0.5ul each |
| FIP/BIP | 2 ul |
| dNTP | 1 ul |
| Bst DNA polymerase Large Fragment | 1 ul |
| ddH2O | 12.5 ul |
| Template | 2 ul |
| Total | 25 ul |

4. Incubate at 65°C for 60-90 minutes
5. Keep the tube at 16°C until the LAMP product is picked up.

## [#](#lateral-flow-assay-lfa) Lateral Flow Assay (LFA)

1. Add 1 ul of detecting probes into the LAMP product and incubate for 37 °C 10 mins.
2. Dilute the reaction solution ten times into a 1.5ml EP tube.
3. Plug the testing strip into the tube and results will be seen within 5 mins. (control line is supposed to be red despite the detecting result otherwise the result is invalid. The testing line being red indicate positive
   results. )

1. [1. 2021 Fudan Protocols](#2021-fudan-protocols)
   1. [1.1 Cell Cryopreservation](#cell-cryopreservation)
   2. [1.2 Cell Recovery](#cell-recovery)
   3. [1.3 PCR](#pcr)
   4. [1.4 Colony PCR](#colony-pcr)
   5. [1.5 Overlap Extension PCR](#overlap-extension-pcr-oe-pcr)
   6. [1.6 Vector PCR](#vector-pcr)
   7. [1.7 Agarose Gel Electrophoresis](#agarose-gel-electrophoresis)
   8. [1.8 DNA Gel Extraction](#dna-gel-extraction)
   9. [1.9 Restriction Enzyme Digestion](#restriction-enzyme-double-digestion)
   10. [1.10 ClonExpress ligation reaction](#clonexpress-ligation-reaction)
   11. [1.11 Plasmid transformation](#plasmid-transformation)
   12. [1.12 Plasmid Miniprep](#plasmid-miniprep-vazyme-fastpure-plasmid-mini-kit)
   13. [1.13 SDS-PAGE](#sds-page)
   14. [1.14 IPTG induction experiment](#iptg-induction-experiment)
   15. [1.15 Growth Curve Measurement](#growth-curve-measurement)
   16. [1.16 Fluorescence Measurement](#fluorescence-intensity-curve-measurement)
   17. [1.17 LAMP](#loop-mediated-isothermal-amplification-lamp)
   18. [1.18 LFA](#lateral-flow-assay-lfa)

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[20-min presentation](https://video.igem.org/w/nwyavmzSGommZxUz9k2nyn)

---

## Written by: Shitao Gong

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# Team:Fudan/Results

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![](https://static.igem.org/mediawiki/2021/6/65/T--Fudan--Candida.png)

# Result

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Updated on 2021-11-18: You might found [this file, which we prepared for the Judging Session,](https://static.igem.org/mediawiki/2021/0/00/T--Fudan--figures4-prepared-for-zoom.pdf) helpful to understand our goals, approaches and what have been achieved.

# [#](#detection-of-calbicans-through-lamp-and-lfa) Detection of *C. albicans* through LAMP and LFA

## [#](#testing-the-availability-of-primers-and-the-dna-in-lamp) **Testing the availability of primers and the DNA in LAMP**

First, to affirm the system could work properly, we started from the very beginning by testing the availability of our primers and the DNA extracted from *Candida albicans* by our instructor following safety rules. As Figure 1
indicates, we got positive results with both agarose gel electrophoresis and LFA.

![pic](https://static.igem.org/mediawiki/2021/1/1e/T--Fudan--result12.jpg)
**Figure 1. positive and specific test of LAMP combined with LFA**. M: DL2000 marker; N:negative control (replacing DNA
of *C. albicans* with \*S. cerevisia); C:\*positive amplification of the DNA of *C. alibicans*

## [#](#identifying-reaction-conditions) **Identifying reaction conditions**

Next, we tried to define the conditions to carry out the detection, including the detection limit, the minimum amount of enzyme required in the reaction, reaction time, and temperature. The reaction time available is above
70 mins. The reaction temperature ranges from 60-65 °C. The minimum amount of enzyme catalyzing LAMP is 2 U/25μl, and the detection limit of *C. albicans* using LAMP is 0.4 pg/μl. 0.4 pg/μl.

![pic](https://static.igem.org/mediawiki/2021/e/e5/T--Fudan--result11.jpg)
**Figure 2. Testing detection conditions.** (a) Testing reaction time. M: marker; Other lanes: different reaction time as indicated.
(b) Testing minimum enzyme requirement to react. M: DL2000 marker; other lanes: different reaction temperature as indicated. (c) Testing reaction temperature. M: DL2000 marker, N: negative
control; other lanes: the different amount of enzyme as indicated. (d) The testing detection limit of LAMP combined with LFA. M: DL2000 marker; other lanes: different template concentration as indicated.

# [#](#function-of-t7-gene-product-circuit) Functions of T7 gene products

In our project, the T7 gene product circuit is used to inhibit the endogenous transcription of *E. coli*, thus enhancing the purity of the protein of interest. To test T7 gp2 and gp5.7, which are respectively known as σ70
inhibitor and σS inhibitor, we must first construct σS promoters for following testing.

**σS promoters verification**

Six σS promoters core region sequences are tested using growth curve measurement and flourescence intensity curve measurement protocol.

![](https://static.igem.org/mediawiki/parts/7/72/T--Fudan--sigmaSpromoters.png)

All of the six sequences are proved to be σS promoters that can function independently. Among them, pdps shows the highest strength as a σS promoter.

Further results can be viewd at the registry page.

http://parts.igem.org/Part:BBa\_K3790014

**Functions of T7 gene product 2**

All the relevant results can be viewd at the registry page.

Please visit:

http://parts.igem.org/Part:BBa\_K3790231

**Functions of T7 gene product 2**

All the relevant results can be viewd at the registry page.

Please visit:

http://parts.igem.org/Part:BBa\_K3790232

# [#](#t7-driven-bst-expression) T7 driven Bst expression

In our project, the T7 expression system (based on the pET52 plasmid) was used to express the large fragment of Bst DNA polymerase I (large fragment), which is required for the isothermal amplification reaction (LAMP to
be specific).

Our Bst (or previously called BstPol) is derived from the DNA polymerase I large fragment in Bacillus stearothermophilus. The large fragment of Bst DNA Polymerase I contains 5' to 3' DNA polymerase activity and strong strand
displacement activity, but lacks 5' to 3' exonuclease activity, ideal for target sequence amplification.

## [#](#construction-of-k3700212) **Construction of K3700212**

We obtained the Bst polymerase large fragment coding DNA optimized for *Escherichia coli* codon from Twist Bioscience, by total synthesis. We have the pET backbone in our laboratory stocks (namely pET16, pET28 and pET52;
all of three shared the same backbone sequence used in our experiments).

We used ClonExpress® Homologous Recombination Kit from Vazyme to perform PCR based cloning, placing Bst (Figure 1) between T7 RBS and T7 terminator. Homology arm primers used are documented at the Design page.

![pic](https://static.igem.org/mediawiki/2021/1/11/T--Fudan--result2.jpg)
**Figure 1. Gel electrophoresis of PCR produts, using Twist synthesis as the template.** The first lane was loaded with DNA ladder,
sizes were marked on the image. The brightest band of 750 bp was about 100 ng, and other bands about 50 ng. The arrowhead marks the target DNA band, approximately 1800bp.

The Bst DNA polymerase I large fragment coding DNA is sized at 1764bp, which is approximately 1800bp after adding homology arms to both ends by PCR reaction. The result (Figure 1) shows a successful amplification of the
DNA of interest. We performed gel extraction of the target region, to obtain pure Bst with homology arms for subsequent reactions.

![pic](https://static.igem.org/mediawiki/2021/e/e0/T--Fudan--result1.jpg)
**Figure 2. Gel electrophoresis of linearized pET bone.** The first lane was loaded with DNA ladder, sizes were marked
on the image. All bands are about 50 ng. The pET plasmid was subjected to loop PCR reaction with homology arm primers in order to linearize the vector. Clone16 and Clone52 were amplified from two different pET stock
DNA templates. The arrowhead marks the target approximately 5200 bp.

Figure 2 indicates that we have obtained two copies of correct sized linearized pET backbone.

After ClonExpress® homologous recombination reaction, Fast-T1 competent bacteria were transformed with reactants, and spread onto LB plates with ampicillin. We picked colony after 16 h culturing plates at 37℃. We further
grow clones in 2 ml LB liquid medium with ampicillin, 37℃ shaking overnight.

Mini-prep was performed the following day, and the resulting plasmids were sent to Sanger sequencing (one correct sequencing result is shown in Figure 3).

![pic](https://static.igem.org/mediawiki/2021/4/44/T--Fudan--result13.jpg)
**Figure 3. Screenshot of one correct sequencing result, only small portion is shown.** This clone was obtained using Clone52 linearized
pET backbone. We sequenced the plasmids from both ends of Bst, and the valid sequences overlapped.

The plasmids containing sequencing verified K3790212 were restriction digested with NcoI and KpnI. The correct digestion pattern is shown in Figure 4.

![pic](https://static.igem.org/mediawiki/2021/1/15/T--Fudan--result3.jpg)
**Figure 4. Gel electrophoresis of plasmids containing K3790212 digested with NcoI and KpnI.** The first lane was loaded with DNA
ladder, sizes were marked on the image. All bands are about 50 ng. Lane labeled with K3790212 is the plasmid DNA without enzymatic digestion. Lane labeled with NcoI/KpnI, is digested. The arrowhead marks the predicted
excised DNA fragment approximately 1800bp.

## [#](#bst-expression) **Bst Expression**

The correct BL21 (DE3) colonies were picked into 2 ml ampicillin LB liquid medium overnight with shaking at 37 ℃.

The following day, the culture was transferred to 2 ml ampicillin LB broth at a 2% volume ratio, and incubation continued at 37 ℃ until reach the logarithmic growth phase of the bacteria (OD600 ~ 0.6). We then started IPTG
induction, for at least 3 hours.

![pic](https://static.igem.org/mediawiki/2021/d/d5/T--Fudan--RESSUP.png)
**Figure 5. Gel electrophoresis of colony PCR of BL21 (DE3) clones.** The first lane was loaded with DNA ladder, sizes were marked
on the image. The brightest band of 750 bp was about 100 ng, and other bands about 50 ng. After obtaining the correct K3790212 recombinant plasmids, we transformed them into BL21 (DE3) competent bacteria for protein
expression. Positive colonies were selected by ampicillin preliminarily and then verified by colony PCR. The arrowhead marks the target approximately 1800bp.

The induced bacterial culture were measure (OD600 absorbance). Some of the bacterial culture was centrifuged to collect a pellet, and a self-made Bst polymerase compatible buffer (BPCB) was added (for formula, see Table
1). After lysis and 4 ℃ 13000rpm 10-min centrifugation, we collected the supernatant (containing Bst polymerase, Figure 6), to perform LAMP reaction. Another portion of the bacterial culture was directly centrifuged
and the pellet was resuspended in 1x SDS sample buffer followed by immediate boiling at 100 ℃ for 5 minutes, will be used for protein SDS-PAGE (Figure 8).

![pic](https://static.igem.org/mediawiki/2021/d/d7/T--Fudan--result5.jpg)
**Figure 6. Compare proteins in the pellet vs. supernatant, after BPCB lysis.**Lane 2 was Bst containing supernant.
We add equal volume of 2x SDS sample buffer. Lane 1 was BPCB insolvable components, from equivalent pellet. As shown in the gel, BPCB solubilizes the bacteria very well, and we do not perform 4 ℃ centrifugation after
seeing this gel.

## [#](#bst-in-bpcb-for-lamp) **Bst in BPCB for LAMP**

The two lanes in Figure 7 show that *Candida albicans* DNA strongly amplified by a lysate of K3790212 containing, IPTG induced, BL21 (DE3) bacteria. The specificity of DNA product was later verified with lateral flow assay
(LFA).

![pic](https://static.igem.org/mediawiki/2021/b/b5/T--Fudan--result4.jpg)
**Figure 7. LAMP reactions using purified Bst or self-made bacterial lysates.** The first lane was loaded with DNA ladder, sizes
were marked on the image. The brightest band of 750 bp was about 100 ng, and other bands about 50 ng. For the last two lanes, we used bacterial lysates from BL21 (DE3) transformed with K37900212, with or without IPTG
induction. Purified Bst represents the purified enzyme.

## [#](#the-initial-test-with-our-gp57-plasmid) **The initial test with our gp5.7 plasmid**

![pic](https://static.igem.org/mediawiki/2021/8/8d/T--Fudan--result6.jpg)
**Figure 8. Compare whole bacteria protein under different induction condition.**BL21 (DE3) bacteria, transformed
with either K3790212 alone, or with both K3790212 and K3790232, were cultured to OD600 ~ 0.6 and then induced as indicated for 3 hours. Whole cell lysates were prepared by directly adding 1x SDS sample buffer into bacteria
pellets, followed by immediate boiling at 100 ℃ for 5 minutes. Lane 5 and Figure 6, was made using a same culture, IPTG induced for 3 hours.

# [#](#fusion-protein-acquisition) Fusion protein acquisition

In our project, we wanted to **make certain modifications to the DNA polymerase of the LAMP reaction**t**o improve its relevant activity and thus the detection limit and precision of the test strips we designed.** In the course of previous studies, we found that the addition of sso7d double-stranded binding protein tended to increase the activity of DNA polymerase, but there is a lack of relevant literature on sso7d to increase
the activity of Bst Pol. Therefore we ventured to hypothesize that sso7d could enhance Bst Pol activity, and we selected three similar double-stranded binding proteins from the same species as sso7d, Sulfolobus solfataricus.
Also, to verify whether single-chain binding proteins also have similar effects, two single-chain binding proteins from Sulfolobus solfataricus and E. coli, respectively, were selected for the experiment. We also considered
that different binding sites of the binding proteins to the polymerase might lead to different results, and therefore we designed whether the binding proteins are attached to the C- or N-terminal end of Bst Pol, respectively.

To construct the fusion proteins, we need to obtain the DNA of Bst Pol and the six binding proteins and ligate Bst Pol and the six binding proteins in the designed positions by homologous recombination.

## [#](#acquisition-of-six-binding-protein-dnas) **Acquisition of six binding protein DNAs**

We have obtained fully synthesized Bst Pol DNA from Twist, and we chose to synthesize the binding proteins by oligo assembly by PCR because of the small number of bases in the six binding proteins.

![pic](https://static.igem.org/mediawiki/2021/2/25/T--Fudan--result7.jpg)
**Figure 9**. Assembled DNA binding proteins, albA1, S.ssb, E.ssb. The first lane was loaded with a **DNA ladder, sizes were marked on the image**.
The brightest band of 750 bp was about 100 ng and other bands about 50 ng. Lanes with correct sized amplified DNA were labeled. After PCR cloning, several bacterial clones were picked, grew into cultures, and sent for
Sanger sequencing. Then, we verified the sequencing results and used the correct ones for further experiments.

![pic](https://static.igem.org/mediawiki/2021/6/69/T--Fudan--result8.jpg)
Figure 10. Assembled DNA binding proteins, DbpA, sso10b, sso7d. The first lane was loaded with a DNA ladder, sizes were marked on
the image. The brightest band of 750 bp was about 100 ng and other bands about 50 ng. Lanes with correct sized amplified DNA were labeled. After PCR cloning, several bacterial clones were picked, grew into cultures,
and sent for Sanger sequencing. Then, we verified the sequencing results and used the correct ones for further experiments.

## [#](#acquisition-of-twelve-fusion-protein-dnas) Acquisition of twelve fusion protein DNAs

As mentioned above, we designed a total of twelve fusion proteins.

|  |  |
| --- | --- |
| Bst Pol 1: Bst-(G2S)3-albA1 | Bst Pol 2: albA1-(G2S)3-Bst |
| Bst Pol 3: Bst-(G2S)3-S.ssb | Bst Pol 4: S.ssb-(G2S)3-Bst |
| Bst Pol 5: Bst-(G2S)3-E.ssb | Bst Pol 6: E.ssb-(G2S)3-Bst |
| Bst Pol 7: Bst-(G2S)3-DbpA | Bst Pol 8: DbpA-(G2S)3-Bst |
| Bst Pol 9: Bst-(G2S)3-Sso10b | Bst Pol 10: Sso10b-(G2S)3-Bst |
| Bst Pol 11: Bst-(G2S)3-Sso7d | Bst Pol 12: Sso7d-(G2S)3-Bst |

To obtain the relevant DNA fragment corresponding to the fusion protein, we added homologous arms to both ends of the DNA fragments of the fusion protein and Bst Pol, thus enabling us to fuse these two DNA fragments into
one complete fragment by overlap PCR.

![pic](https://static.igem.org/mediawiki/2021/d/d6/T--Fudan--result9.jpg)
**Figure 11. Assembled fusion proteins, Bst Pol 1, Bst Pol 2, Bst Pol 3, Bst Pol 4, Bst Pol 5, Bst Pol 6.** The first
lane was loaded with a DNA ladder, sizes were marked on the image. The brightest band of 750 bp was about 100 ng and other bands about 50 ng. Lanes with correct sized amplified DNA were labeled. After PCR cloning, several
bacterial clones were picked, grew into cultures, and sent for Sanger sequencing. Then, we verified the sequencing results and used the correct ones for further experiments.

![pic](https://static.igem.org/mediawiki/2021/a/a9/T--Fudan--result10.jpg)
**Figure 12. Assembled fusion proteins, Bst Pol 7, Bst Pol 9, Bst Pol 11.** The first lane was loaded with a DNA ladder, sizes were
marked on the image. The brightest band of 750 bp was about 100 ng and other bands about 50 ng. Lanes with correct sized amplified DNA were labeled. After PCR cloning, several bacterial clones were picked, grew into
cultures, and sent for Sanger sequencing. Then, we verified the sequencing results and used the correct ones for further experiments.

![pic](https://static.igem.org/mediawiki/2021/6/63/T--Fudan--add.png)
**Figure 13**. Assembled fusion proteins, Bst Pol 8, Bst Pol 10, Bst Pol 12. The first lane was loaded with a **DNA ladder, sizes were marked on the image.** The brightest band of 750 bp was about 100 ng and other bands about 50 ng. Lanes with correct sized amplified DNA were labeled. After PCR cloning, several bacterial clones were picked, grew into cultures, and sent for
Sanger sequencing. Then, we verified the sequencing results and used the correct ones for further experiments.

The result (Figure 3/4/5) shows a successful amplification of the DNA of interest. We performed gel extraction of the target region, obtain pure fusion protein DNA with homology arms for subsequent reactions. After ClonExpress®
homologous recombination reaction, Fast-T1 competent bacteria were transformed with reactants and spread onto LB plates with ampicillin. We picked colony after 16 h culturing plates at 37℃. We further grow clones in
2 ml LB liquid medium with kanamycin, 37℃ shaking overnight.

Mini-prep was performed the following day, and the resulting plasmids were sent to Sanger sequencing, our synthesized DNA sequence was confirmed to be correct.

1. [1. LAMP and LFA](#detection-of-calbicans-through-lamp-and-lfa)
   1. [1.1. Testing the availability of primers and the DNA in LAMP](#testing-the-availability-of-primers-and-the-dna-in-lamp)
   2. [1.2. Identifying reaction conditions](#identifying-reaction-conditions)
2. [2. Functions of T7 gene products](#function-of-t7-gene-product-circuit)
3. [3. T7 driven Bst expression](#t7-driven-bst-expression)
   1. [3.1. Construction of K3700212](#construction-of-k3700212)
   2. [3.2. Bst Expression](#bst-expression)
   3. [3.3. Bst in BPCB for LAMP](#bst-in-bpcb-for-lamp)
   4. [3.4. The initial test with our gp5.7 plasmid](#the-initial-test-with-our-gp57-plasmid)
4. [4. Fusion protein acquisition](#fusion-protein-acquisition)
   1. [4.1. Acquisition of six binding protein DNAs](#acquisition-of-six-binding-protein-dnas)
   2. [4.2. Acquisition of twelve fusion protein DNAs](#acquisition-of-twelve-fusion-protein-dnas)

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---

## Lead by: Xin Lan