iGEM 2021 — Team NOFLS_YZ

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# Team:NOFLS YZ/Notebook

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# Notebook

# DRY LAB NOTEBOK

![](https://static.igem.org/mediawiki/2021/6/61/T--NOFLS_YZ--img_notebook_1.jpg)

## 7/29

* -Made some icebreakers to help team members get known to each other and build friendship and team
  soul
* -iGEM opening report meeting
* -Introduction to IBD

![](https://static.igem.org/mediawiki/2021/8/8f/T--NOFLS_YZ--img_notebook_2.jpg)

## 8/12

* -Determination of team name
* -Team logo design
* -Team caption selection
* -Selection of team products
* -Online expert interview

![](https://static.igem.org/mediawiki/2021/5/52/T--NOFLS_YZ--img_notebook_3.jpg)

## 8/13

* -Online survey production

## 8/14

* -WeChat tweet writing

## 09/19

* -Introduction of the subject
* -Completed the project PPT production
* -Discussion of analytical questionnaire online
* -Design of expert interview questions
* -Discussion of sales of team derivatives'

![](https://static.igem.org/mediawiki/2021/c/c3/T--NOFLS_YZ--img_notebook_4.jpg)

## 09/20

* -Message broadcasting on WeChat
* -Pricing of team products
* -Team collaboration
* -Team member' self introduction writing

![](https://static.igem.org/mediawiki/2021/7/76/T--NOFLS_YZ--img_notebook_5.jpg)

## 10/1

* -Team photo shooting

## 10/2

* -Collaboration Meetups

![](https://static.igem.org/mediawiki/2021/3/35/T--NOFLS_YZ--img_notebook_6.jpg)

## 10/3

* -Overall review of the experiment

## 10/4

* -Wiki material writing

## 10/5

* - Expert Interview

![](https://static.igem.org/mediawiki/2021/6/67/T--NOFLS_YZ--img_notebook_7.jpg)

# WET LAB NOTEBOOK

![](https://static.igem.org/mediawiki/2021/3/3f/T--NOFLS_YZ--img_notebook_8.jpg)

## 2021.9.11

* 1. Bacteria expressed pUC57-amilGFP/pSU2718-p15A activated.
* 2. Plasmid transformation into Top 10 competent cell

  Plasmid A: pUC57-PyeaR\_HrpR

  Plasmid B: pUC57-PttB344\_HrpS\_PJ23105\_ttrR

  Plasmid C: pUC57(Kan)-PhrpL\_amilGFP\_IL10\_PJ23104\_ttrS

## 2021.9.12

* 1. Extracted plasmid pUC57-amilGFP and pSU2718-p15A
* 2. Enzyme digestion (Plasmid pSU2718-p15A)
* 3. DNA agarose gel electrophoresis identification
* 4. DNA agarose gel reavers and purification

## 2021.9.13

* 1. Extracted plasmid pUC57-PyeaR\_HrpR, pUC57-PttB344\_HrpS\_PJ23105\_ttrR and
  pUC57(Kan)-PhrpL\_amilGFP\_IL10\_PJ23104\_ttrS
* 2. PCR to get part 2 (PttB344\_HrpS\_PJ23105\_ttrR)
* 3. DNA agarose gel electrophoresis identification
* 4. DNA agarose gel reavers and purification
* 5. T4 ligase link digested plasmid pSU2718-p15A and part2 PttB344\_HrpS\_PJ23105\_ttrR
* 6. Recombinant plasmid transformation

## 2021.9.14

* -Use micropipette tips to pick a single colony from the plate (from day 9.13), put it in LB
  liquid medium.

![](https://static.igem.org/mediawiki/2021/9/97/T--NOFLS_YZ--img_notebook_9.jpg)

## 2021.9.15

* 1. Plasmid pSU2718-Part2 extraction
* 2. Enzyme digestion identification
* 3. Plasmid was sent to sequence

## 2021.9.17

* Correct plasmid pSU2718-Part2 extraction

## 2021.9.18

* 1. Enzyme digestion (Plasmid pSU2718-Part2)
* 2. PCR to get part 1 (PyeaR\_HrpR)
* 3. DNA agarose gel electrophoresis identification
* 4. DNA agarose gel reavers and purification
* 5. T4 ligase link digested plasmid pSU2718-Part2 and part1 PyeaR\_HrpR
* 6. Recombinant plasmid transformation

![](https://static.igem.org/mediawiki/2021/0/01/T--NOFLS_YZ--img_notebook_10.jpg)

## 2021.9.19

* Bacteria PCR identification

![](https://static.igem.org/mediawiki/2021/f/fd/T--NOFLS_YZ--img_notebook_11.jpg)
![](https://static.igem.org/mediawiki/2021/0/0a/T--NOFLS_YZ--img_notebook_12.jpg)

## 2021.9.20

* 1. Plasmid pSU2718-Part2-Part1extraction
* 2. Enzyme digestion identification
* 3. Positive plasmid was sent to sequence

![](https://static.igem.org/mediawiki/2021/f/f6/T--NOFLS_YZ--img_notebook_13.jpg)

## 2021.9.22

* Plasmid transformation (pUC57(Kan)-PhrpL\_amilGFP\_IL10\_PJ23104\_ttrS) into Top 10 cell.

## 2021.9.24

* 1. Prepare Top10/pUC57(Kan)-PhrpL\_amilGFP\_IL10\_PJ23104\_ttrS competent cell；
* 2. Plasmid (pSU2718-Part2-Part1) transformation into
  Top10/pUC57(Kan)-PhrpL\_amilGFP\_IL10\_PJ23104\_ttrS competent cell

## 2021.9.25

* Use micropipette tips to pick a single colony from the plate (from day 9.24), put it in LB liquid
  medium.

## 2021.9.26

* Plasmid extraction.

## 2021.10.1-2021.10.7

* Function tests

## CONTACT INFO

WeChat Official Account: *IBD Distinguishers*

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# Team:NOFLS YZ/Engineering

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# Engineering

# Background

Inflammatory bowel disease (IBD) is a chronic intestinal inflammatory disease of unknown etiology,
including ulcerative colitis (CD) and Crohn’s disease (CD). This chronic disease, which is prone to
repeated deterioration, currently lacks unified diagnostic and treatment standards, and is posing a
great threat to public health. Drug therapy (anti-inflammatory drugs) is the preferred treatment for
IBD. However, studies in the past 10 years have found that 30-50% of IBD patients do not respond to
anti-TNF treatment. In addition, after long-term use of anti-inflammatory drugs, the patient's
intestinal microbial status changes over time, and the effect may be lost due to drug resistance.
Therefore, we need to seek help from other treatments for IBD.

At present, some researchers have developed engineered bacteria that respond to biomarkers of intestinal
inflammation, demonstrating the feasibility of using synthetic biology to design engineered bacteria in
the field of IBD treatment.

# Design

Colonic bacteria can produce a large amount of genetically toxic hydrogen sulfide (H2S) during
metabolism, and the lumen mucosa can protect itself from H2S by converting H2S
into thiosulfate
(S2O32-). The sulfate-reducing bacteria in the patient's intestines
produce ROS (reactive oxygen
species) to convert S2O32- into tetrathionate/tetrathionate
(Tetrathionate, S4O62-). In addition,
inflammation of the intestine can induce high-level expression of nitric oxide synthase (iNOS) and
increase the production of NO. NO is converted to nitrate (NO3-). Therefore, both
S2O32-/ S4O62- and
NO3- can be used as biomarkers for the development of biosensors for intestinal inflammation.

We can build Boolean logic circuits to combine thiosulfate/tetrasulfate and nitrate biosensors to
optimize performance, improve sensor accuracy and enhance robustness, and optimize intestinal
inflammation detection (Figure 1). When and only when
S2O32-/S4O62- and
NO3- are present at the same
time, the fluorescent gene is expressed (Figure 1). The fluorescent gene can be replaced with a gene
that produce the interleukin-10 (anti-inflammatory factor) for the treatment of IBD.

![](https://static.igem.org/mediawiki/2021/a/aa/T--NOFLS_YZ--img_engineering_1.jpg)
Figure 1. Boolean gate logic circuit/AND circuit construction.

# Build

There are three composite parts were constructed using pUC57 or pSU2718 vector as backbone (Figure
2).

![](https://static.igem.org/mediawiki/2021/b/b2/T--NOFLS_YZ--img_engineering_2.jpg)
Figure 2. A. PyeaR\_HrpR, sensing NO3- to release the substance R; B. PttB344\_HrpS\_PJ23105\_ttrR, sensing S4O62- to release the substance S; C. PhrpL\_amilGFP\_IL10\_PJ23104\_ttrS.

# Test

## (1) Function of S2O32-/S4O62-和NO3- biosensor

*Escherichia coli* was transformed with pUC57-PhrpL\_amilGFP\_IL10\_PJ23104\_ttrS, and or pSU2718-
PyeaR\_HrpR\_
PttB344\_HrpS\_PJ23105\_ttrR. We can see that there was no fluorescence in Sample 3 (the report part)
(Figure 3). According to the result of Sample 3 and Sample 4 (Figure 3), we could infer that the design
works, since the pUC57-PhrpL\_amilGFP\_IL10\_PJ23104\_ttrS didn’t respond when PyeaR\_HrpR and
PttB344\_HrpS\_PJ23105\_ttrR were not present.

![](https://static.igem.org/mediawiki/2021/4/4c/T--NOFLS_YZ--img_engineering_3.jpg)
Figure 3. Sample 1: Negative Control (*E. coli*); Sample 2: Positive Control (*E. coli*/amilGFP); Sample 3: *E. coli*/ pUC57-PhrpL\_amilGFP\_IL10\_PJ23104\_ttrS; Sample 4: E.coli/ pSU2718- PyeaR\_HrpR-PttB344\_HrpS\_PJ23105\_ttrR + pUC57-PhrpL\_amilGFP\_IL10\_PJ23104\_ttrS.

## (2) Model build

In order analyze the relationship between OD600 and the fluorescence intensity, we built a model by
MATLAB (Table 1).

Table 1. Model of OD600 and the fluorescence intensity
![](https://static.igem.org/mediawiki/2021/5/58/T--NOFLS_YZ--img_engineering_4.jpg)

After roughly analyze the scatter plots, we chose to use the quadratic polynomial equation to adapt to
our data:

![](https://static.igem.org/mediawiki/2021/7/76/T--NOFLS_YZ--img_engineering_5.jpg)

Coding we used in MATLAB is given below:

```
clear;clc;
od0=[0.6 0.8 1];
intensity1=[1356 5822 6611];
intensity2=[1221 4709 6189];
intensity3=[1578 6700 5927];
intensity0=(intensity1+intensity2+intensity3)/3;
p=polyfit(od0,intensity0,2);
od=[0.6:0.01:1];
intensity=polyval(p,od);
plot(od,intensity,'LineWidth',2)
ymax=max(intensity)
i=find(intensity==max(intensity));
xmax=od(i)
hold on
plot(od0,intensity0,'r*','LineWidth',2)
plot(xmax,ymax,'g*','LineWidth',2)
hold off
```

After calculation, the constants of this model are listed below:

Table 2.Constants of model
![](https://static.igem.org/mediawiki/2021/1/17/T--NOFLS_YZ--img_engineering_6.jpg)

Therefore the final model would be:

![](https://static.igem.org/mediawiki/2021/b/bc/T--NOFLS_YZ--img_engineering_7.jpg)

![](https://static.igem.org/mediawiki/2021/a/af/T--NOFLS_YZ--img_engineering_8.jpg)
Figure 4. The fitting curve of the model f(x)=-48250x2+89343x-34851

According to the fitting curve (Figure 4), it indicates that the optimal condition for our biosensor to
detect IBD related compounds, would be the time when OD600 reaches 0.93 around. This result could be
also served as a reference for more performance tests in the future after we complete the improvement of
our biosensor genetic design.

## CONTACT INFO

WeChat Official Account: *IBD Distinguishers*

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# Team:NOFLS YZ/Experiments

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# Experiments

# PCR

-A gene of interest can be amplified from the genome of any animal, plant, bacterial plasmid containing
the gene of interest as a template for PCR

-Experimental material

![](https://static.igem.org/mediawiki/2021/8/8e/T--NOFLS_YZ--img_experiments_1.png)

* system: 50μl
* -mix at each step
* -template: bacteria (plasmid containing)

# Electrophoresis

Fabrication of agarose gels

Experimental Materials:

![](https://static.igem.org/mediawiki/2021/f/fe/T--NOFLS_YZ--img_experiments_2.png)

* -DNA is negatively charged and will be electrophoresed from negative to the positive pole
* -The nucleic acid dye will bind to the DNA and after beating the blue light the fluorescence
  (appears red)
* -Marker: usually drops on the extreme edge of the lattice
* Marker is a mixture of double-stranded DNA standards of different lengths, by which the DNA
  length of the sample can be known by comparison
* -Loading: to settle the sample, glycerol containing may increase the sample density so that
  sedimentation into the spotting wells prevents samples from fluctuating out

Recovery of DNA

1. Take a clean scalpel to excise the DNA fragment per 100 mg
2. Determine the weight of the gel slice and transfer it 50 °C to a clean tube.
3. Add Buffer B to agarose gel.
4. Incubate the sample at 50 °C until the gel slice is completely dissolved.
5. Transfer the solution into the adsorption column and centrifuge 8000g for 30 seconds. Drain the
   liquid from the collection tube.
6. Add 500 μL Wash solution, centrifuge 9000g for 30 seconds, and pour ou the liquid in the
   collection tube.
7. Repeat step 6 once.
8. Centrifuge the empty adsorption column at 9000g for 1 min
9. Place the adsorption column into a new 1.5 mL micro centrifuge tube. Add 15 µL Elution Buffer and
   incubate at room temperature (25°C) for 1 min.
10. Centrifuge for 1 minute at 9000 g

Nucleic acid detection

-Detection of DNA concentration

# Plasmid purification

-Kit Method

-Experimental operation steps:

1. The bacteria were removed by centrifugation from the medium, and the underlying liquid was
   decanted

   After decanting, the remaining medium was blotted onto filter paper and blotted

   Keeping bacteria: keep the remaining bacteria for later experiments
2. Add reagents to the kit, and buffer 123 in that order (ingredients mainly ions, glucose,
   etc.)

   The most important of buffer 1 is that there are RNases
3. Shake the tube and allow the buffer to mix with the enzyme

   Note be sure to mix completely, without directly affecting subsequent operations
4. Lyase 250 μ L

   Buffer 2: alkaline solution (SDS and NaOH)

   SDH: disruption of cell membrane

   NaOH: Base

   Lysis, disruption of cell membrane / cell structure of bacteria

   Denaturation, opening the duplex structure to single stranded

   Mix by inversion (solution consists of turbidity → clear)
5. Add buffer 3

   Ingredients: CH3COOH, CH3COOK

   Action: neutralization, alkaline solution, renaturation (single stranded → double stranded)

   Mix by shaking gently (6-8 times)

   All that remains is a few flocculent impurities (cellular debris)
6. Centrifuge at 120000 g for 10 min to obtain the supernatant (750 mL with the goal of avoiding the
   pellet, change the tip of the gun every time)
7. Centrifuge the supernatant and pour off the following solution again
8. Wash by adding 500 μL wash solution
9. Centrifuge (1 min at 10000 rpm), take the solid
10. Repeat step 8.9
11. Centrifuge empty column(2 min at 10000 rpm)

    Action: remove 75% ethanol remaining from wash solution
12. Allow to stand at room temperature for 2 min
13. Put the column into a clean 1.5mL tube, add 15μL EB buffer and centrifuge for 1min.

# Enzymatic digestion

-Material:

![](https://static.igem.org/mediawiki/2021/7/77/T--NOFLS_YZ--img_experiments_3.png)

Put it in 37 ℃ for 1h

-Note:

If the digestion time is too long, there will be non-specific cutting, which may be cut into several
sections

# LB Culture Medium Preparation

-Material:

* Yeast extract 5 g/L
* Tryptone 10 g/L
* NaCl 10 g/L
* Agar\* 1.5% of total mass
* ddH2O Desired volume

\*Agar is only necessary when LB culture medium is prepared as solid agar gel.

1. Sterilization

Sterilize at 121 °C for 30 minutes.

2. KANA antibiotics

Concentration of KANA antibiotics in solution is 50 mg/μL.

Functional concentration of KANA antibiotics is 50 μg/μL.

# Plasmid Transformation

-The plasmid after ligation was aspirated and placed into competent cells

Because competent cells are very fragile, they need to be cryopreserved and manipulated
gently

-Experimental manipulations:

1. Place on ice competent cells with the plasmid for 30min

Make sufficient contact of competent cells with the plasmid

2.42℃ heat shock

Allow competent cells to crack, allowing plasmids to enter the cell

3. Place on ice for 2 min

Allowing the bacteria to regain activity

4. Add 1 mL LB liquid medium without resistance

Repair resistance (let resistance genes express)

5. The plates were incubated at 37 ° C for 1 h

6. Centrifugation

Fix the bacteria to the bottom

# Colony PCR

-Principle: take the bacteria as the template and use the primer amplification (PCR) of the target gene.
Only the successfully connected recombinant plasmid can be copied

-Material:

![](https://static.igem.org/mediawiki/2021/4/42/T--NOFLS_YZ--img_experiments_4.png)

# Plasmid Extraction

-Kit Method

-Experimental operation steps:

1. - Pick up monoclonal colonies on the plate, inoculate in 10 mL LB liquid medium, and cultivate in
incubator for 12 hours to 16 hours at 250 rpm.

2. - Add 500μL P1 to the CP3 adsorption column, centrifugalize at 12000 rpm for 1 minute to activate
the silicon membrane.

3. - Take 1mL bacterial liquid. Centrifugate for 1 minute at 12000 rpm, and remove the
supernatant.

4. - Add 250μL P1 and resuspend the precipitate.

5. - Add 250μL P2 and gently overturn the tube for 6 to 8 times.

6. - Add 350μL P3. Gently overturn the tube for 6 to 8 times, and centrifugate for 10 minutes at
12000 rpm after the white precipitates form.

7. - Collect the supernatant to the column. Centrifugate for 30 to 60 seconds at 12000 rpm, and
remove the liquid.

8. - Add 600μL PW. Centrifugate for 30 to 60 seconds at 12000 rpm. Remove the liquid, and repeat the
process.

9. - Centrifugate at 12000 rpm for 2 minutes.

10. - Put the column into a new centrifuge tube, add 100μL EB. Keep the tube in room temperature for
2 minutes, and centrifugate at 12000 rpm for 2 minutes.

# Function test

Reaction solution preparation

-NaNO3(1 mol/L) diluted 100 times →10 mmol/L

-Di(dithioperoxo)sulfuricacid/tetrathiosulfonate（0.1 mol/L）diluted 40 times → 2.5mmol/L

-Composition of each tube configured:

5ml medium + 5μL sodium nitrate + 125 μL sodium tetrathiosulfonate

-Place the reagents needed for detection onto the microplate reader

The signal intensity has a direct relationship with the volume of liquid

-The solution was divided into 5 parts at 0 h, 1 h, 2 h, 3 h, 4 h

-The 0 h solution was replaced by blank medium, nitrate, sodium tetrathiosulfonate (because it would not
be detected by the microplate reader)

GNP detection:

-The solution was divided into 5 parts,

either of the two kind of salt above was added

only NaNO3 was added

only tetrathiosulfonate was added

both sodium nitrate and sodium tetrasulfide sulfonate were added

-Light irradiation (high energy, blue) at 485 nm wavelength was used

-Emit long wavelength light (low energy, green)

## CONTACT INFO

WeChat Official Account: *IBD Distinguishers*

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# Team:NOFLS YZ/Results

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# Results

# Recombinant Plasmid Construction

![](https://static.igem.org/mediawiki/2021/2/2a/T--NOFLS_YZ--img_results_1.jpg)
Figure 1. The electrophoresis results of enzyme digestion and PCR.

Lane 1 and 2: Plasmid pSU2718-p15A digested by Xba1 and BamH1.

Lane 3: Part 2, PttB344\_HrpS\_PJ23105\_ttrR, got by PCR method with size of 2060bp.

![](https://static.igem.org/mediawiki/2021/4/4e/T--NOFLS_YZ--img_results_2.jpg)
Figure 2 E-coil having the desired pSU2718-part2 (Left) and control (Right).

![](https://static.igem.org/mediawiki/2021/9/98/T--NOFLS_YZ--img_results_3.jpg)
Figure 3. The electrophoresis result of enzyme digestion identification.

Lane 1: Plasmid pSU2718-p15A digested by EcoR1.

Lane 2 to 5: Recombinant plasmid pSU2718-part2 digested by EcoRI. We got two bands with size of 2803bp
and 1544bp.

The results show that we got the correct plasmid. And the plasmid was sent to sequence.

![](https://static.igem.org/mediawiki/2021/9/9a/T--NOFLS_YZ--img_results_4.jpg)
Figure 4 The result of sequencing for plasmid pSU2718-part2.

Sequencing feedback shows we have obtained the correct plasmids which is consistent with their DNA
profiles.

![](https://static.igem.org/mediawiki/2021/6/63/T--NOFLS_YZ--img_results_5.jpg)
Figure 5 The electrophoresis results of enzyme digestion (Middle) and PCR (Right).

Middle:

Lane 1 and 2: Plasmid pSU2718-part2 digested by HindIII.

Right:

Lane 1: Part 1, PyeaR\_HrpR, got by PCR method with size of 1293bp.

![](https://static.igem.org/mediawiki/2021/5/50/T--NOFLS_YZ--img_results_6.jpg)
Figure 6 E-coil having the desired pSU2718-part2-part1 (Left) and control (Right).

![](https://static.igem.org/mediawiki/2021/7/71/T--NOFLS_YZ--img_results_7.jpg)
Figure 7 The electrophoresis result of enzyme digestion.

Lane 1: Recombinant plasmid pSU2718-part2-part1 without enzyme digestion.

Lane 2: Recombinant plasmid pSU2718-part2-part1 digested by EcoR1.

The result shows that we got the correct plasmid. And the plasmid was sent to sequence.

![](https://static.igem.org/mediawiki/2021/d/d3/T--NOFLS_YZ--img_results_8.jpg)
Figure 8 The result of sequencing for plasmid pSU2718-part2-part1

Sequencing feedback shows we have obtained the correct plasmids which is consistent with their DNA
profiles.

# Function Test Results

* Part1: **PyeaR\_HrpR**, sensing NO3- to release the substance
  R;
* Part2: **PttB344\_HrpS\_PJ23105\_ttrR**, sensing
  S4O62- to release the
  substance S;
* Part3: **PhrpL\_amilGFP\_IL10\_PJ23104\_ttrS**, will release fluorescence
  if sensing the substances, R
  and S released from Part 1(HrpR) and Part 2 (HrpS) both.

![](https://static.igem.org/mediawiki/2021/e/e4/T--NOFLS_YZ--img_results_9.jpg)
Genetic Construction of IBD Distinguisher

## Attempt 1

![](https://static.igem.org/mediawiki/2021/0/08/T--NOFLS_YZ--img_proof_of_concept_2.jpg)
Graph 1.

* Sample 1: NO3- + S4O62-
* Sample 2: S4O62-
* Sample 3: NO3-
* Sample 4: Blank Control

As shown above, there is obvious fluorescence in sample 1 and sample 2; for sample 3 and sample 4, there
also a slight fluorescence produced in the tubes. In order to scientifically quantify the result, we
used ELIASA to read the accurate fluorescence intensity, respectively.

Table 1.
![](https://static.igem.org/mediawiki/2021/f/fd/T--NOFLS_YZ--img_proof_of_concept_3.jpg)

![](https://static.igem.org/mediawiki/2021/a/a3/T--NOFLS_YZ--img_proof_of_concept_4.jpg)
Graph 2. Histogram of the fluorescence intensity of samples in Table 1

According to the histogram above, we can see that Sample 1 (NO3-+
S4O62-) presents obvious higher
fluorescence intensity than the rest three samples which means our design working. But we do realize
that Sample 2 (S4O62-) also presents fluorescence than the blank
control which confused us a lot.
According to our design, if we only add S4O62- without NO3-,
this AND gate wouldn’t allow to pass into
the reporter (Part 3) as there is a closed door of the NO3- sensor (Part 1). Therefore, we
thought there
was something wrong in Part 1.

In order to figure out the reason, we did several literature research and find out that the potential
causes of Part 1’s failure may be that we didn’t build the NarX-NarL two-component regulator (Graph 3),
which shall be the first one to sense nitrate and that is why Part 1 didn’t work fine as we
expected.

![](https://static.igem.org/mediawiki/2021/3/34/T--NOFLS_YZ--img_proof_of_concept_5.jpg)
Graph 3. Genetic construction design map
Ref. Woo, Seung-Gyun, et al. "A designed whole-cell biosensor for live diagnosis of gut inflammation through nitrate sensing." Biosensors and Bioelectronics 168 (2020): 112523.

## Attempt 2

![](https://static.igem.org/mediawiki/2021/4/4a/T--NOFLS_YZ--img_proof_of_concept_6.jpg)
Graph 4.

* Sample 1: Negative Control (E. coli)
* Sample 2: Positive Control (E. coli/amilGFp)
* Sample 3: E. coli/pUC-57\_Part 3
* Sample 4: E.coli/pSU2718-Part 1\_Part 2+pUC-57\_Part 3

In order to further analyze the performance of Part 2, we also designed the control groups as showing
above where Sample 1 and Sample 2 were presented as the comparison of the fluorescence phenomenon. We
can see that there was no fluorescence in Sample 3 (the report part, Part 3). According to the result of
Sample 3 and Sample 4 (*Graph 4*), we could infer that the AND
gate design works (**HrpR\_****HrpS**, **PhrpL**) as the Part 3 didn’t respond when there is no Part 1 and Part
2.

Of course, we also repeated the experiments and measured the fluorescence intensity by ELIASA. The data
is given below.

Table 2.
![](https://static.igem.org/mediawiki/2021/a/aa/T--NOFLS_YZ--img_proof_of_concept_7.jpg)

![](https://static.igem.org/mediawiki/2021/9/91/T--NOFLS_YZ--img_proof_of_concept_8.jpg)
Graph 5. Histograms of the fluorescence intensity of samples in Table 2

![](https://static.igem.org/mediawiki/2021/1/18/T--NOFLS_YZ--img_proof_of_concept_9.jpg)
Graph 6. Trend Contrast of the fluorescence intensity between that under OD600=0.8 and that under OD600=1

According to the trend in Graph 6, the fluorescence intensity of pUC57-Part 3 presents the similar level
as that of blank control and group NO3-, which means the “gate keeper” promoter, **PhrpL** works well and
this conclusion also back up the conclusion that the AND gate design works well (**HrpR\_****HrpS**, **PhrpL**).

As the group S4O62- did present higher fluorescence intensity than that
of group NO3- whenever OD600
equals to 0.8 or 1, **it indicates that Part 2 and Part 3 both works** well as Part 2 did show “green
light”
to the tetrathionate to finally “light” the Part 3.

# Future Plan

So far we have ensured that Part 2, Part 3 and also the AND gate design work, we need to improve the
design of Part 1 which is used to sense nitrate so that we could fully complete the whole biosensor
construction to diagnose IBD.

Besides, we also have concerns about the biosensor carrier, E. coli. Firstly, we need ensure that our
engineered bacteria would not be immediately degraded or eliminated by other enteric microorganism.
Secondly, E.coli may have adverse effects such as septicemia in patients with immune deficiency to the
gut microbiome. In the future, safer bacteria like lactobacillus should be found as carrier receptors
for transformation.

## CONTACT INFO

WeChat Official Account: *IBD Distinguishers*