iGEM 2024 — Team hwfla-beijing

{% extends "layout.html" %}
{% block title %}Notebook{% endblock %}
{% block lead %}Document the dates you worked on your project. This should be a detailed account of the work done each
day for your project.{% endblock %}
{% block page\_content %}

![](https://static.igem.wiki/teams/5526/desc-banner.png)

{{ self.title() }}

* 2024.8.19
* 2024.8.20
* 2024.8.21
* 2024.8.22
* 2024.8.23
* 2024.8.24
* 2024.8.25
* 2024.8.27
* 2024.8.28
* 2024.8.29-9.15

2024.8.19

### Participants

Li Jialiang, Wu Siyuan, Liu Lanxin, Li Tianyi

### Goal

* Prepare LB medium (1L) for bacterial culture
* Prepare 100mg/ml Ampicillin
* Prepare 0.05mol/L CaCl2
* Inoculate Azurin template strain, anti-PD-L1 template strain, plactate1 template
  strain, plactate2 template strain

### Content

- 1.Prepare LB medium (1L) for bacterial culture

LB liquid:

| Material | Dosage |
| --- | --- |
| Yeast extract | 5g |
| Trypsin | 10g |
| Sodium chloride | 10g |

LB Solid

| Material | Dosage |
| --- | --- |
| LB liquid | 100ml |
| Agar powder | 1.5g |

Sterilize at high temperature 121℃, 15min

- Prepare a 10ml Ampicilin solution with a final concentration of 100μg/mL
- Prepare 0.05mol/L CaCl2

  | Material | Dosage |
  | --- | --- |
  | CaCl2 | 0.28g |
  | Water | 100ml |

  Mix and sterilise at high temperature121℃，15min
- Prepare 0.05mol/L CaCl2 with glycerin

  | Material | Dosage |
  | --- | --- |
  | CaCl2 | 0.28g |
  | 15% glycerin | 15ml |
  | Water | 100ml |

  Mix and sterilize at high temperature121℃, 15min
- Inoculate 4 kinds of template bacteria solution

  | Promoter template | Target gene template |
  | --- | --- |
  | Plactate1 (LldR) | Azurin |
  | Plactate2 (LldR-new) | anti-PD-L1 |

  Get four combinations using them：Plactate1-Azurin、Plactate1-anti-PD-L1、Plactate2-Azurin、Plactate2-anti-PD-L1

  Using a benchtop to inoculate 5ml of LB、5μL of Ampicilinand 50ul of bacteria solution

2024.8.20

### Participants

Li Jialiang, Wu Siyuan, Liu Lanxin, Li Tianyi

### Goal

* Plasmid extraction
* PCR amplification
* Use agarose gel electrophoresis to detect whether DNA was obtained
* DNA recovery

### Content

* Plasmid was extracted：

  | Material | Dosage |
  | --- | --- |
  | Solution I: SP1 bacterial suspension | 250μL |
  | Solution II: SP2 cell lysis buffer | 250μL |
  | Solution III: SP3 neutralization solution | 350μL |
  | Wash solution | 750μL |

  1-5ml of bacterial culture (P1, P2, Azurin, anti-PD-L1) was centrifuged at 12000rpm for 1min, and the
  supernatant was absorbed

  + Add 250μL solution to the centrifugation tube containing the bacterial precipitation and suspend the
    bacterial cell precipitation thoroughly using a vortex oscillator
  + Add 250μL solution Ⅱ into the centrifuge tube and gently turn it up and down 6-8 times to fully crack
    the bacteria. At this time, the bacteria solution will become clear and viscous
  + Add 350u solution III into the centrifuge tube and turn it up 6-8 times to mix well. The white, fluffy
    precipitate will appear at this time. Centrifuge at 12000rpm for 10min and transfer the supernatant to
    another clean centrifuge tube
  + Add 0.35 times the volume of anhydrous ethanol and mix well
  + Add the supernatant into the adsorption column, leave at room temperature for 2min, centrifuge at
    12000rpm for 1min, drain the waste liquid in the collection tube, and put the adsorption column back
    into the collection tube
  + Add 750μL bleach solution to the adsorption column, centrifuge at 12000rpm for 1min, and put the
    adsorption column into the collection tube
  + Add 700L bleaching solution to the adsorption column, centrifuge at 12000rpm for 1min, and put the
    adsorption column into the collection tube
  + Centrifuge at 12000rpm for 2min and place the adsorption column at room temperature or 50℃ for several
    minutes
  + Place the adsorption column into a clean centrifuge tube, drop 50μL eluent preheated in a water bath at
    65℃ into the center of the adsorption film, place at room temperature for 2min, and centrifuge at
    12000rpm for 1min
  + The eluent obtained was re-added into the adsorption column, placed at room temperature for 2min, and
    centrifuged at 12000rpm for 1min
* PCR amplification (target gene amplification)：

  | Material | Dosage |
  | --- | --- |
  | Target gene (P1, P2, anti-PD-L1, Azurin) | 2 μL |
  | 2\*Mix (dNTP, DNA polymerase, Buffer, magnesium ion) | 25 μL |
  | Primer (F) | 2 μL |
  | Primer (R) | 2 μL |
  | ddH2O | 19 μL |

  + Set up the PCR reaction program
  + Set up the PCR reaction program
  Program:

  1.Pre degeneration： Double chain to single chain 95°C，5min

  2.dimorphism:95°C，30s

  3.annealing：55°C，30s （Repeat 1-3 30 times）

  4. extending：72°C，30s for gene /2min for plasmid

  5. 72 ℃，10 min

  6. 4°C，∞

  + Initiate the program
  + Agarose gel electrophoresis was used to detect whether DNA was amplified
  + Prepare50mL agarose gel

  | Material | Dosage |
  | --- | --- |
  | Agarose powder | 0.5 g |
  | TAE buffer solution | 50 mL |
  | Nucleic acid dye | 3 μL |

  + Melt agarose powder and TAE buffer at high temperature and add 3μL nucleic acid dye
  + Install the electrophoresis tank
  + Gently pour the electrophoresis solution cooled to 60 °C onto the horizontal plate of the
    electrophoresis tank
  + After it has solidified, add the electrophoresis buffer to the bath and pull out the comb
  + Add the mixture to the sample tank
  + Connect one end of the sample hole to the negative electrode and the other to the positive electrode.
    Turn on the power supply, adjust the voltage to 180V, and electrophoresis for 20min
  + The gel was removed and observed under an ultraviolet lamp. The DNA bands with red fluorescence were
    found
* DNA gel extraction

  | Material | Dosage |
  | --- | --- |
  | Glue solution | 300 μL / 0.1 g DNA gel block |
  | Bleach wash solution | 600 μL |

  + After agar-gel electrophoresis, a single DNA strip of interest is cut from the agar-gel and weighed
  + Add 600μL sol solution to the glue block and place in a water bath at 55℃ for 10 min. During the
    process, gently turn the centrifuge tube up and down continuously to ensure that the glue block is fully
    dissolved
  + The solution was added to an adsorption column and centrifuged at 12000pm for 60 seconds
  + Add 600μL bleaching solution to the adsorption column and centrifuge at 12000rpm for 1min, discard the
    waste liquid, and put the adsorption column into the collection tube
  + Add 600μL bleach solution into the adsorption column, centrifuge 12000rpm for 1min, discard the waste
    liquid, and put the adsorption column into the collection tube
  + Centrifuge at 12000rpm for 2min, remove the bleach solution as much as possible, and place the
    adsorption column open at room temperature or 50℃ in a temperature box
  + Place the adsorption column into a clean centrifuge tube, drop the appropriate amount of eluent
    preheated in a water bath at 65℃ into the center of the adsorption film, place at room temperature for
    2min, and centrifuge at 12000rpm for l min
  + DNA products stored at -20℃

### Result

![](https://static.igem.wiki/teams/5526/notebook/1.jpg)

A. plactate1, plactate2 plasmid skeleton amplification, the size are 3150bp and 3800bp
plactate1-Azurin, plactate2-Azurin, plactate1-anti-PD-L1, plactate2-anti-PD-L1, target gene amplification size
is 550bp

2024.8.21

### Participants

Li Jialiang, Wu Siyuan, Liu Lanxin, Li Tianyi, Wu Peiran

### Goal

* The target gene sfGFP was obtained by PCR
* Recombinant plasmid was constructed by homologous recombination
* The recombinant product transformed Escherichia coli BL21

### Content

* The target gene sfGFP was obtained by PCR
  PCR system
  Target gene (P1, P2, sfGFP): 2μL
  2\*Mix (dNTP, DNA polymerase, Buffer, magnesium ion): 25 uL
  Primer (F/R): F:2μL R:2μL
  ddH2O: 19μL set the PCR reaction program
  Procedure: Pre degeneration: double chain to single chain 95°C，5min
  Degeneration：95°C，30s
  Annealing：55°C，30s （Repeat 30 times）
  Extending：72°C，30s for GFP /2min for plasmid
  72 ℃，10 min
  4°C，∞
  agarose gel formula

  | Material | Dosage |
  | --- | --- |
  | Total | 50 mL |
  | Agarose | 0.5 g |
  | 1\*TAE | 50 mL |

  After 2 minutes of high-fire melting, add 3μL of dye and pour it into the mold.
* Homologous recombination
  the amount of DNA required for the recombination reaction was calculated according to the formula, and the
  amount of each component added was not less than 1μl
  Prepare the following reaction system on the ice:

  | Component | Recombination reaction |
  | --- | --- |
  | Linearized carrier | 2 μL |
  | Target gene segment | 1 μL |
  | 2 × ClonExpress Mix | 5 μL |
  | ddH2O | 2 μL |

  Use a pipette to gently suck and mix (do not shake and mix), and briefly centrifuge to collect the reaction
  liquid to the bottom of the tube. 50℃, 15 min; Reduce to 4 ° C or cool immediately on ice
* Transformation of recombinant products
  Thawing chemoreceptor cells for cloning on ice
  Add 10 μl recombinant product into 100 μL receptive cells, gently mix the tube wall (do not shake and mix),
  and leave it on ice for 30 min.
  After the 42℃ water bath is heated for 45 seconds, it is immediately placed on ice to cool for 3 min.
  Add 900 μL LB liquid medium (without antibiotics) and shake the bacteria at 37℃ for 1 h (rotational speed
  200 rpm).
  Preheat the corresponding resistant LB solid medium plate in a 37℃ incubator.
  Centrifuge at 5000 rpm (2,500 × g) for 5 min and discard 900 μL supernatant. The bacteria sediment was
  suspended with the remaining medium and lightly coated with a sterile coating stick on a plate containing
  Amp resistance.
  Culture inversely in an incubator at 37℃ for 12 h.

### Result

![](https://static.igem.wiki/teams/5526/notebook/2.jpg)

Left：plactate1, placate2 Plasmid skeleton amplification size are 3150bp and 3800bp
Right：plactate1-sfGFP, plactate2-sfGFP amplification size are both 750bp

2024.8.22

### Participants

Li Jialiang, Wu Siyuan, Liu Lanxin, Li Tianyi

### Goal

* Colony PCR verification
* Inoculate the correct single colony

### Content

* PCR amplification (colony expansion):
  The target genes anti-PD-L1, Azurin, and sfGFP were amplified by PCR
  Material dose
  DNA Template: 6 recombinant plasmid colonies (anti-PD-L1, Azurin, GFP)
  2\*Mix (dNTP, DNA polymerase, Buffer, magnesium ion) 10μL
  (F/R): F:1μL R:1μL
  ddH2O: 8μL
  Set up the PCR reaction program
  + Predegeneration: double chain to single chain95°C，5min
  + degeneration：95°C，30s
  + Annealing：55°C，30s
  + Extending：72 °C，1min
    Step 2-4 repeat 30 times
  + 72°C, 10 min
  + 6. 4°C, ∞
* Prepare50mL agarose gel
  Material dose
  Agarose powder 1g
  TAE buffer 100mL
  Nucleic acid dye 18μL
  Melt agarose powder and TAE buffer at high temperature and add 6μL nucleic acid dye
  \* Install the electrophoresis tank
  \* Gently pour the electrophoresis solution cooled to 60°C onto the horizontal plate of the electrophoresis
  tank
  \* After it solidifies, add the electrophoresis buffer to the electrophoresis tank and then pull out the
  comb
  \* Add the mixture to the sample tank
  \* One end of the electrophoresis point sample hole is connected to the negative electrode, and the other
  end is connected to the positive electrode; turn on the power supply,
  \* Adjust voltage to 180V, electrophoresis 20min
  \* Remove the gel and observe it under an ultraviolet lamp. The red fluorescent bands are DNA bands
* Verify the correct colony by inoculation
  plactate1-Azurin, plactate2-Azurin, plactate1-anti-PD-L1, plactate2-anti-PD-L1
  4mL LB solution + correct colony +4uL Ampicilin was cultured at 37 ° C for 12h

### Result

plactate1-sfGFP, plactate2-sfGFP, Recombinant plasmid validation
A: Colony PCR validation diagram
B: Plate colony map
C: Sequencing diagram

![](https://static.igem.wiki/teams/5526/notebook/3.jpg)

plactate1-Azurin, plactate2-Azurin, plactate1-anti-PD-L1, plactate2-anti-PD-L1
Recombinant plasmid validation
A: Colony PCR validation diagram
B: Plate colony map
C: Sequencing diagram

![](https://static.igem.wiki/teams/5526/notebook/4.jpg)

2024.8.23

### Participants

Li Jialiang, Wu Siyuan, Liu Lanxin, Li Tianyi, Wu Peiran

### Goal

* Plasmid extraction (plactate1-Azurin, plactate1-anti-PD-L1, plactate2-Azurin, plactate2-anti-PD-L1)
* Inoculate ECN 1917 to make ECN 1917 receptive state
* Heat shock transformation of recombinant plasmid to ECN 1917
* Expanded culture of Plactae1-GFP and Plactate2-GFP with different lactic acid-induced expression (cultured
  at 18℃ for 20h)

### Content

* Plasmid extraction (same steps as 2024.8.20)
* Inoculate ECN 1917 to make ECN 1917 receptive state
  + Transfer ECN1917 bacteria solution cultured overnight at 1% inoculation rate (37℃, 220rpm, culture for
    2.5h to 0.3 for OD600)
  + bacterial harvest: Centrifuge at 4℃ 5000rpm for 5 min after ice bath
  + Wash precipitate in cold deionized water and centrifuge at 4°C5000rpm for 5 min
  + precipitation Add 2ml of 0.05mol/l CaCl2-15% glycerol mixture to cold, gently blow away, and take an ice
    bath of 0.05mol/l CACL2-
  + 15% glycerol mixture for 5 minutes
  + The receptive cell suspension was formed by adding 2ml of cold 0.05mol/l CaCl2-15% glycerol mixture and
    gently blowing it away. Divided into 50-100μL small parts, stored at -70℃
* Heat shock conversion (same steps as 2024.8.21)
  plactate1-sfGFP and plactate2-sfGFP were cultured with different concentrations of lactic acid (0mM, 2mM,
  5mM, 10mM) to induce the expression of GFP fluorescent protein

### Result

![](https://static.igem.wiki/teams/5526/notebook/5.jpg)

Converted to ECN 1917
A: Colony PCR validation diagram in ECN1917
B: Plate colony map

2024.8.24

### Participants

Wu Siyuan, Liu Lanxin, Li Tianyi, Wu Peiran

### Goal

* Colony PCR verified whether the recombinant plasmid was transformed into EcN 1917
* Inoculate the correct single colony
* The expression of fluorescent protein sfGFP was detected

### Content

* PCR amplification (colony amplification) :
  PCR amplification of target genes P1, P2, anti-PD-LI, Azurin in EcN(550bp)
  Material dose
  1 colony (P1, P2, anti-PD-L1, Azurin, GFP)
  2\*Mix (dNTP, DNA polymerase, Buffer, magnesium ion) 10μL
  (F/R) : F:1μL R:1μL
  ddH2O 8μL
  • Set up PCR reaction procedures
  1. Predenaturation: double chain to single chain 95°C, 5min
  2.Denaturation: 95°C, 30s
  3. Annealing: 55°C, 30s
  4. Extension: 72 °C, 30s
  2-4 Repeat 30 times
  5. 72°C, 10 min
  6. 4°C, ∞
* Configure 100mL agarose gel
  Material dose
  Agarose powder 1g
  TAE buffer 100mL
  Nucleic acid dye 6μL
  Melt agarose powder and TAE buffer at high temperature and add 6μL nucleic acid dye
  \* Install the electrophoresis tank
  \* Gently pour the electrophoresis solution cooled to 60°C onto the horizontal plate of the electrophoresis
  tank
  \* After it has solidified, add the electrophoresis buffer to the electrophoresis tank and then pull out
  Comb
  \* Add the mixture to the sample tank
  \* One end of the electrophoresis point sample hole is connected to the negative electrode, the other end is
  connected to the positive electrode; turn on the power supply,
  \* Adjust voltage to 180V, electrophoresis 20min
  \*Remove the gel and observe it under an ultraviolet lamp. The red fluorescent bands are DNA bands
* GFP fluorescence determination
  + Fluorescence microscope detection, sfGFP luminescence.
  + ELISA reading (black 96-well plate, 470nm excitation light, 509nm emission light, reading value;
    Transparent 96-well plate, OD600 reading value; Former reading/latter reading = relative fluorescence
    intensity)
* Verify the correct colony by inoculation
  plactate1-Azurin, plactate2-Azurin, plactate1-anti-PD-L1, plactate2-anti-PD-L1 in EcN
  4mL LB solution + correct colony +4μL Amp+ was cultured at 37 ° C for 12h

  ![](https://static.igem.wiki/teams/5526/notebook/6.jpg)

  The luminescence of sfGFP was observed by a fluorescence microscope.

2024.8.25

### Participants

Li Jialiang, Wu Siyuan, Liu Lanxin, Li Tianyi, Wu Peiran

### Goal

* The 4 EcN strains successfully transformed by expanded culture were cultured at 37 degrees for 2-4h.
* Set different culture OD and other lactic acid induction concentrations and induce culture at 37 degrees for more than 10h
* Configure different concentrations of lactic acid, 0mM, 2mM, 5mM, 10mM

### Content

* The 4 EcN strains successfully transformed by expanded culture were cultured at 37 degrees for 2-4h.
  The correct 4 plasmids were transformed into Escherichia coli ECN, the correct clone and the correct clone, and the correct clone was inoculated into seed liquid.
  Then, the seed solution was transferred to amplify E.coli until OD=0.5-0.6.
* Set different culture OD and other lactic acid induction concentrations and induce culture at 37℃ for more than 10h
  Lactic acid was added for induction (the final concentration was 0 25 10mM), the bacterial solution and supernatant were collected for about 10 hours (the ECN clone was selected to culture overnight, and the ratio of 1 to 100 was inoculated into the medium containing lactic acid), and the protein expression was subsequently detected.

### Result

![](https://static.igem.wiki/teams/5526/notebook/7.jpg)

2024.8.27

### Participants

Li Jialiang, Wu Siyuan, Liu Lanxin, Li Tianyi, Wu Peiran

### Goal

* Culture four strains to induce expression
* Broke the protein
* Make SDS-PAGE protein measuring gel

### Content

* Cultivate Four Strains and Induce Expression

  To induce the expression of four proteins, we used 0mM, 2mM, 5mM, and 10mM lactic acid, respectively. Obtain the bacterial suspensions cultured overnight and centrifuge them at 4°C, 5000rpm, for 5 minutes to separate the supernatant from the precipitate. Discard the supernatant and retain the precipitate. Resuspend the residue in 10 ml of PBS buffer. Repeat the centrifugation process at 4°C, 5000rpm for 5 minutes, discard the supernatant, retain the residue, and resuspend it in 10ml of PBS buffer.
* Protein Disruption

  Perform ultrasonic disruption at 100w with a 3-second on and 3-second off cycle for 10 minutes. Ensure the entire process is conducted on ice, with 16 sets. After disruption, centrifuge to collect the supernatant, discard the residue, and measure the protein concentration.
* Preparation of SDS-PAGE Gel for Protein Analysis

  Assemble the required equipment and apparatus. Then, prepare the upper stacking and lower resolving gel using a 10% PAGE gel solution.

2024.8.28

### Participants

Li Jialiang, Wu Siyuan, Liu Lanxin, Li Tianyi, Wu Peiran

### Goal

* Prepare protein sample
* Test the expression of protein

### Content

* Prepare protein sample
  The bacterial supernatant was added to 5xSDS-PAGE protein loading buffer and thoroughly mixed. Heat the mixture at 100℃ for10min, then 12000rpm centrifuge for 2min. The supernatant was subjected to electrophoresis by 10%SDS-PAGE.
* Test the expression of protein
  It is necessary to ensure that the protein content of the samples is consistent and that the size of target proteins in the electrophoresis results are consistent through Coomassie bright blue staining.

2024.8.29-9.15

### Participants

Li Jialiang, Wu Siyuan, Liu Lanxin, Li Tianyi, Wu Peiran

### Goal

* Write experimental data processing and wiki writing

### Content

Complete the experimental data processing and wiki.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a technical aspect of your project by going through at least one iteration of the engineering design cycle. This achievement should be distinct from your Contribution for Bronze.{% endblock %}
{% block page\_content %}

![](https://static.igem.wiki/teams/5526/desc-banner.png)

{{ self.title() }}

* Overview
* Cycle 1: Plldr-sfGFP (BBa\_K822002)
* Cycle 2: Plldr(new)-sfGFP (BBa\_K5526003)
* Cycle 3: Plldr-anti PD-L1 (BBa\_K5526004)
* Cycle 4: Plldr(new)-anti PD-L1 (BBa\_K5526006)
* Cycle 5: Plldr-Azurin (BBa\_K5526005)
* Cycle 6: Plldr(new)-Azurin (BBa\_K5526007)
* Other Test
* Summary

Overview

Cancer metastasis makes it difficult to treat. Systemic treatment for metastatic cancer often results in serious side effects. Although the development of large molecule targeted drugs, immunomodulatory drugs, and cell therapies has dramatically improved the therapeutic effect of cancers. The most kind of advanced cancers still cannot be cured. Oncolytic viruses and bacteria are another promising cancer treatment method. Among them, anaerobic bacteria are natural tumor targeting. Our project genetically modified probiotics to express anti-tumor proteins to enhance their anti-cancer effects. In order to improve the tumor specificity of the engineered bacteria, we constructed an expression system induced by lactic acid, which only expresses and releases anti-tumor protein when entering the high lactic acid environment inside the tumor. The goal of our products is to achieve targeted clearance of tumor cells.

![](https://static.igem.wiki/teams/5526/eng/1.png)

Figure 1. The engineering design schematic diagram.

Cycle 1: Plldr-sfGFP (BBa\_K822002)

**Design 1:**

In the plasmid Plldr\_sfGFP (referred to as plactate1-sfGFP) we constructed, we combined Plldr(BBa\_K822000), sfGFP(BBa\_K4716993), and pUC57-mini(BBa\_K3983004) together to form Plldr-sfGFP(BBa\_K822002).

Plldr is a lactic acid promoter that will be activated under a high lactic acid concentration, which is a trait of tumor areas. sfGFP will be transcribed and form fluorescence protein. pUC57 is the skeleton of the plasmid. We’d also add Amp+ into it to ensure only the EcN1917 with the correct plasmid will grow. plactate1-sfGFP is the plasmid that can be activated and produce fluorescent proteins by a high lactic acid concentration.

![](https://static.igem.wiki/teams/5526/eng/2.png)

Figure 2. The plasmid map of Plldr\_sfGFP

**Build 1:**

We applied PCR on the genes sfGFP(750bp) and pUC57- Plldr (3150bp); we used agarose gel electrophoresis to check the length of our PCR production to ensure we succeeded. The results showed that the pUC57- Plldr(plactate1) had a length of 3150 bp, and the sfGFP had a length of 750 bp (Figure 3).

![](https://static.igem.wiki/teams/5526/eng/3.jpg)

Figure 3. The identification of PCR production by agarose gel electrophoresis
Left: The graph shows that plactate 1 has a length of 3150 bp.
Right: the graph shows p1-sfGFP has a length of 750bp.

We first used homologous recombination to combine sfGFP with the lldr promoter, forming the Plldr-sfGFP (plactate1-sfGFP) construct. We then performed a heat shock conversion to make BL21(DE3) cells sensitive to frequent changes in temperature, alternating between high and low temperatures to facilitate the uptake of plasmids of BL21(DE3). After heat shock, we injected the plasmids into BL21(DE3) cells and grew them on an Amp+ medium, ensuring that only bacteria containing the plasmids would survive. As expected, bacterial colonies grew on the petri dishes, indicating successful plasmid uptake. To further confirm the presence of the desired plasmid, we performed a colony PCR directly from the colonies on the plate. This allowed us to amplify the specific region of the plasmid containing the Plldr-sfGFP(plactate1-sfGFP) construct. Figure 4 shows the PCR results were positive, indicating that the colonies contained the correct plasmid. Finally, we recycled the plasmids and sent them for sequencing at a bio company to ensure the correct sequence. The sequencing results confirmed that the plasmids were indeed the ones we wanted, with the correct sequence and no mutations.

![](https://static.igem.wiki/teams/5526/eng/4.jpg)

Figure 4. PCR identification of plactate1-sfGFP plasmid.
A. The graph shows the length of p1-sfGFP is 750bp.
B. The graph shows the flora growing in a petri dish.
C. The graph shows the sequencing measured by the bio company.

**Test 1:**

We analyzed the fluorescence intensity of sfGFP produced using two different approaches:

Fluorescence microscope: We first used the fluorescence microscope to test the lightness of the sfGFP. This is a qualitative test to visually observe under what concentration of lactic acid the lightness of sfGFP will reach the highest. The microscope provided qualitative data, showing that the fluorescence intensity reaches the highest when the lactic acid concentration is 5mM, indicating that the Plldr-sfGFP (plactate1-sfGFP) construct was functioning as intended (Figure 5).

![](https://static.igem.wiki/teams/5526/eng/5.jpg)

Figure 5. The microscopic images of bacteria under white light and fluorescence.
The graph shows that sfGFP reaches the highest fluorescence intensity at 5mM of lactic acid concentration.

Fluorescent Microplate Reader: We then applied a quantitative test to the sfGFP using the fluorescent microplate reader. The microplate reader provided precise numerical data on the fluorescence emitted by the cells; we analyzed the data and drew a graph based on it. From this analysis, we concluded that the fluorescence intensity of sfGFP was highest at a lactic acid concentration of 5mM, confirming the optimal response of the construct to this concentration (Figure 6).

![](https://static.igem.wiki/teams/5526/eng/6.jpg)

Figure 6. Bacterial fluorescence intensity at different lactate concentrations. The graph we drew after analyzing the data from the fluorescent microplate reader also shows the highest light intensity at 5mM of lactic acid concentration.

**Learn 1:**

This plasmid was constructed from a comparison with Plldr(new)-sfGFP to show whether the improvement on the new Plldr is functional. The experiment would be successful if the Plldr(new)-sfGFP got a higher light intensity than Plldr-sfGFP.

Cycle 2: Plldr(new)-sfGFP (BBa\_K5526003)

**Design 2:**

In the plasmid Plldr(new)-sfGFP (BBa\_K5526003) we constructed, we combined Plldr-new (BBa\_K5526001), sfGFP (BBa\_K4716993), and pUC57-mini (BBa\_K3983004) together to form Plldr(new)-sfGFP (plactate2-sfGFP).

Plldr(new) is a lactic acid promoter that will be activated under a high lactic acid concentration, which is the trait of tumor areas. It gets several improvements to the original promoter. It is more accurate and will not get inhibited within low oxygen concentrations. sfGFP will be transcribed and form fluorescence protein. pUC57 is the skeleton of the plasmid. We’d also add Amp+ into it to ensure only the EcN1917 with the correct plasmid will grow. Plldr(new)-sfGFP is the plasmid that can be activated and produce fluorescent proteins by a high lactic acid concentration. Plldr(new)-sfGFP is more sensitive and precise when increasing lactic acid concentration. In addition, it will not be limited within low oxygen concentration, like what the original one did instead.

![](https://static.igem.wiki/teams/5526/eng/7.png)

Figure 7. The plasmid map of Plldr(new)-sfGFP

**Build 2:**

We applied PCR on the genes sfGFP(750bp) and pUC57- Plldr (new)(3800bp); we used agarose gel electrophoresis to check the length of our PCR production to ensure we succeeded. The result is that the plactate 2 got a length of 3800bp, and the sfGFP got a length of 750bp.

![](https://static.igem.wiki/teams/5526/eng/8.jpg)

Figure 8. The identification of PCR production by agarose gel electrophoresis
Left: The graph shows that plactate 2 has a length of 3800 bp.
Right: the graph shows p2-sfGFP has a length of 750bp.

We first used homologous recombination to combine sfGFP with the new lldr promoter, forming the Plldr(new)-sfGFP (plactate 2-sfGFP) construct. We then performed a heat shock conversion to make BL21(DE3) cells sensitive to frequent changes in temperature, alternating between high and low temperatures to facilitate the uptake of plasmids of BL21(DE3). After heat shock, we injected the plasmids into BL21(DE3) cells and grew them on an Amp+ medium, ensuring that only bacteria containing the plasmids would survive. As expected, bacterial colonies grew on the petri dishes, indicating successful plasmid uptake. To further confirm the presence of the desired plasmid, we performed a colony PCR directly from the colonies on the plate. This allowed us to amplify the specific region of the plasmid containing the Plldr(new)-sfGFP (plactate 2-sfGFP) construct. Figure 9 shows the PCR results were positive, indicating that the colonies contained the correct plasmid. Finally, we recycled the plasmids and sent them for sequencing at a bio company to ensure the correct sequence. The sequencing results confirmed that the plasmids were indeed the ones we wanted, with the correct sequence and no mutations.

![](https://static.igem.wiki/teams/5526/eng/9.jpg)

Figure 9. PCR identification of plactate2-sfGFP plasmid.
A. The graph shows the length of p2-sfGFP is 750bp.
B. The graph shows the flora growing in a petri dish.
C. The graph shows the sequencing measured by the bio company.

**Test 2:**

We analyzed the fluorescence intensity of sfGFP produced using two different approaches:

Fluorescence microscope: We first used the fluorescence microscope to test the lightness of the sfGFP. This is a qualitative test to visually observe under what concentration of lactic acid the lightness of sfGFP will reach the highest. The microscope provided qualitative data, showing that the fluorescence intensity reaches the highest when the lactic acid concentration is 5mM, indicating that the Plldr(new)-sfGFP (plactate 2-sfGFP) construct was functioning as intended (Figure 10).

![](https://static.igem.wiki/teams/5526/eng/10.jpg)

Figure 10. The microscopic images of bacteria under white light and fluorescence.
The graph shows that the sfGFP reaches the highest fluorescence intensity at 5 mM for lactic acid concentration.

Fluorescent Microplate Reader: We then applied a quantitative test to the sfGFP using the fluorescent microplate reader. The microplate reader provided precise numerical data on the fluorescence emitted by the cells; we analyzed the data and drew a graph based on it. From this analysis, we concluded that the fluorescence intensity of sfGFP was highest at a lactic acid concentration of 5mM, confirming the optimal response of the construct to this concentration (Figure 11).

![](https://static.igem.wiki/teams/5526/eng/11.jpg)

Figure 11. Bacterial fluorescence intensity at different lactate concentrations. The graph we drew after analyzing the data from the fluorescent microplate reader also shows the highest light intensity at 5mM of lactic acid concentration.

**Learn 2:**

This plasmid has been constructed from a comparison with Plldr-sfGFP to show whether the improvement on the new Plldr is functional. The experiment would be successful if the Plldr(new)-sfGFP got a higher light intensity than Plldr-sfGFP.

Cycle 3: Plldr-anti PD-L1(BBa\_K5526004)

**Design 3:**

In the plasmid plactate1-antiPD-L1(BBa\_K5526004) we constructed, we combined Plldr(BBa\_K822000), antiPD-L1(BBa\_K5526008), and pUC57-mini(BBa\_K3983004) together to form Plldr-antiPD-L1(plactate 1- antiPD-L1).

Plldr is a lactic acid promoter that will be activated under a high lactic acid concentration. antiPD-L1 is a set gene that encodes an immunomodulatory protein, which can activate the human body’s immune system and kill the tumor cells. pUC57 is the skeleton of the plasmid. Plldr-antiPD-L1 is the plasmid that will be activated and produce an immunomodulatory drug by high lactic acid concentration.

![](https://static.igem.wiki/teams/5526/eng/12.png)

Figure 12. The plasmid map of Plldr- anti PD-L1

**Build 3:**

We applied PCR on the genes antiPD-L1(372bp) and pUC57-plldr(3150bp); we used agarose gel electrophoresis to check the length of our PCR production to ensure we succeeded. The result is that the pUC57-plldr (plactate 1) got a length of 3150bp, and the antiPD-L1 got a length of 372bp.

![](https://static.igem.wiki/teams/5526/eng/13.jpg)

Figure 13. The identification of PCR production by agarose gel electrophoresis
Left: The graph shows that plactate 1 has a length of 3150 bp.
Right: The graph shows that antiPD-L1 has a length of 372bp.

We first used homologous recombination to combine anti PD-L1 with the lldr promoter, forming the Plldr-anti PD-L1. We then performed a heat shock conversion to make DH5α cells sensitive to frequent changes in temperature, alternating between high and low temperatures to facilitate the uptake of plasmids of DH5α. After heat shock, we injected the plasmids into DH5α cells and grew them on an Amp+ medium, ensuring that only bacteria containing the plasmids would survive. As expected, bacterial colonies grew on the petri dishes, indicating successful plasmid uptake. To further confirm the presence of the desired plasmid, we performed a colony PCR directly from the colonies on the plate. This allowed us to amplify the specific region of the plasmid containing the Plldr-antiPD-L1(plactate 1- antiPD-L1) construct. Figure 14 shows the PCR results were positive, indicating that the colonies contained the correct plasmid. Finally, we recycled the plasmids and sent them for sequencing at a bio company to ensure the correct sequence. The sequencing results confirmed that the plasmids were indeed the ones we wanted, with the correct sequence and no mutations.

![](https://static.igem.wiki/teams/5526/eng/14.jpg)

Figure 14. PCR identification of plactate1-antiPD-L1 plasmid.
A. The graph shows the length of p1-antiPD-L1 is 372bp.
The graph shows the flora growing in a petri dish.
C. The graph shows the sequencing measured by the bio company.

**Test 3:**

We used alkaline lysis to extract plasmids (plactate1-Anti-PD-L1) from bacterial cultures. Next, we converted the recombinant plasmid to EcN1917 competent by heat shock transformation. PCR was used to verify whether the plasmid was transformed into EcN1917. The results are shown in Figure 15-A. The colony PCR results were detected by agarose gel electrophoresis, and the amplified band was 550bp, which was consistent with the target band, indicating that the four plasmids were successfully transformed into EcN1917.

![](https://static.igem.wiki/teams/5526/eng/15.jpg)

Figure 15. PCR identification of EcN1917 transformants.
A. The PCR production of the plactate1-Anti-PD-L1 transformants by agarose gel electrophoresis.
B. The graph shows the flora growing in a petri dish.

We grow the bacteria holding the plasmids under different OD values (0.3, 0.6, 0.8,1.0) and lactic acid concentrations (0mM, 2mM, 5mM, 10mM). We used the nanodrop to detect protein concentration and created a graph of four different bacteria’s protein expression. We conclude that the protein concentration is highest when OD600 equals 0.6, and the lactic acid concentration is 5mM. We used SDS-PAGE to ensure we got the proteins we wanted.

![](https://static.igem.wiki/teams/5526/eng/16.jpg)

Figure 16. The effects of bacterial concentration and lactic acid concentration on the expression of plactate1-anti PD-L1.

**Learn 3:**

This plasmid Plldr-antiPD-L1(plactate1-antiPD-L1) was constructed to form a comparison with Plldr(new)-antiPD-L1. If the concentration of proteins produced by the Plldr(new)-antiPD-L1 is higher than Plldr-antiPD-L1(plactate1-antiPD-L1), this can show that improving the new Plldr is useful.

Cycle 4: Plldr(new)-anti PD-L1(BBa\_K5526006)

**Design 4:**

In the plasmid Plldr(new)-antiPD-L1 (BBa\_K5526006) we construct, we combined Plldr-new (BBa\_K5526001), antiPD-L1 (BBa\_K5526008), and pUC57-mini (BBa\_K3983004) together to form Plldr(new)-antiPD-L1 (plactate2-antiPD-L1).

Plldr-new gets several improvements to the original promoter. It is more accurate and will not get inhibited within low oxygen concentrations. antiPD-L1 is a set gene that encodes an immunomodulatory protein, which can activate the human body’s immune system and kill the tumor cells. pUC57-mini is the skeleton of the plasmid. Plldr(new)-antiPD-L1 (plactate2-antiPD-L1) is the plasmid that will be activated and produce an immunomodulatory drug by high lactic acid concentration. It is more sensitive and precise when increasing lactic acid concentration. In addition, it will not be limited within low oxygen concentration, like what the original one did instead.

![](https://static.igem.wiki/teams/5526/eng/17.png)

Figure 17. The plasmid map of Plldr(new)-anti PD-L1

**Build 4:**

We applied PCR on the genes antiPD-L1(372bp) and pUC57-plldr-new(3800bp). We used agarose gel electrophoresis to check the length of our PCR production to ensure we succeeded. The result is that the pUC57-plldr-new (plactate 2) got a length of 3800bp, and the antiPD-L1 got a length of 372bp.

![](https://static.igem.wiki/teams/5526/eng/18.jpg)

Figure 18. The identification of PCR production by agarose gel electrophoresis
Left: The graph shows that plactate 2 has a length of 3800 bp.
Right: The graph shows that p2-antiPD-L1 has a length of 372bp.

We first used homologous recombination to combine antiPD-L1 with the new lldr promoter, forming the Plldr(new)-antiPD-L1 (plactate2-antiPD-L1). We then performed a heat shock conversion to make DH5α cells sensitive to frequent changes in temperature, alternating between high and low temperatures to facilitate the uptake of plasmids of DH5α. After heat shock, we injected the plasmids into DH5α cells and grew them on an Amp+ medium, ensuring that only bacteria containing the plasmids would survive. As expected, bacterial colonies grew on the petri dishes, indicating successful plasmid uptake. To further confirm the presence of the desired plasmid, we performed a colony PCR directly from the colonies on the plate. This allowed us to amplify the specific region of the plasmid containing the plactate2-antiPD-L1 construct. Figure 18 shows the PCR results were positive, indicating that the colonies contained the correct plasmid. Finally, we recycled the plasmids and sent them for sequencing at a bio company to ensure the correct sequence. The sequencing results confirmed that the plasmids were indeed the ones we wanted, with the correct sequence and no mutations.

![](https://static.igem.wiki/teams/5526/eng/19.jpg)

Figure 19. PCR identification of plactate2-antiPD-L1 plasmid.
A. The graph shows the length of p2-antiPD-L1 is 372bp.
B. The graph shows the flora growing in a petri dish.
C. The graph shows the sequencing measured by the bio company.

**Test 4:**

We used alkaline lysis to extract plasmids (plactate2-Anti-PD-L1) from bacterial cultures. Next, we converted the recombinant plasmid to EcN1917 competent by heat shock transformation. PCR was used to verify whether the plasmid was transformed into EcN1917. The results are shown in Figure 20-A. The colony PCR results were detected by agarose gel electrophoresis, and the amplified band was 550bp, which was consistent with the target band, indicating that the four plasmids were successfully transformed into EcN1917.

![](https://static.igem.wiki/teams/5526/eng/20.jpg)

Figure 20. PCR identification of EcN1917 transformants.
A. The PCR production of the plactate2-Anti-PD-L1 transformants by agarose gel electrophoresis.
B. The graph shows the flora growing in a petri dish.

We grow the bacteria holding the plasmids under different OD values (0.3, 0.6, 0.8,1.0) and lactic acid concentrations (0mM, 2mM, 5mM, 10mM). We used the nanodrop to detect protein concentration and created a graph of four different bacteria’s protein expression. We conclude that the protein concentration is highest when OD600 equals 0.6, and the lactic acid concentration is 5mM. We used SDS-PAGE to ensure we got the proteins we wanted.

![](https://static.igem.wiki/teams/5526/eng/21.jpg)

Figure 21. The effects of bacterial concentration and lactic acid concentration on the expression of plactate2-antiPD-L1.

**Learn 4:**

This plasmid was constructed from a comparison of Plldr-antiPD-L1. If the concentration of proteins produced by the Plldr(new)-antiPD-L1(plactate2-antiPD-L1) is higher than Plldr-antiPD-L1, this can show that improving the new Plldr is useful.

Cycle 5: Plldr-Azurin (BBa\_K5526005)

**Design 5:**

In the plasmid Plldr-Azurin (BBa\_K5526005) we constructed, we combined Plldr (BBa\_K822000), Azurin (BBa\_K5526000), and pUC57-mini (BBa\_K3983004) to form Plldr-Azurin (plactate 1-Azurin).

Plldr is a lactic acid promoter that will be activated under a high lactic acid concentration. Azurin is a gene that encodes a chemical drug, which can release some medicines and substances to kill the tumor cells. pUC57-mini is the skeleton of the plasmid. Plldr-Azurin is the plasmid that will be activated and produce a toxic protein with a high lactic acid concentration.

![](https://static.igem.wiki/teams/5526/eng/22.png)

Figure 22. The plasmid map of Plldr- Azurin.

**Build 5:**

We applied PCR on the genes Azurin(444bp) and pUC57-plldr (3150bp); we used agarose gel electrophoresis to check the length of our PCR production to ensure we succeeded. The result shows that the pUC57-plldr (plactate 1) has a length of 3150bp, and the Azurin has a length of 444bp.

![](https://static.igem.wiki/teams/5526/eng/23.jpg)

Figure 23. The identification of PCR production by agarose gel electrophoresis
Left: The graph shows that plactate 1 has a length of 3150 bp.
Right: The graph shows p1-Azurin has a length of 444bp.

We first used homologous recombination to combine Azurin with the lldr promoter, forming the Plldr-Azurin (plactate1-Azurin). We then performed a heat shock conversion to make DH5α cells sensitive to frequent changes in temperature, alternating between high and low temperatures to facilitate the uptake of plasmids of DH5α. After heat shock, we injected the plasmids into DH5α cells and grew them on an Amp+ medium, ensuring that only bacteria containing the plasmids would survive. As expected, bacterial colonies grew on the petri dishes, indicating successful plasmid uptake. To further confirm the presence of the desired plasmid, we performed a colony PCR directly from the colonies on the plate. This allowed us to amplify the specific region of the plasmid containing the Plldr-Azurin (plactate1-Azurin) construct. Figure 22 shows the PCR results were positive, indicating that the colonies contained the correct plasmid. Finally, we recycled the plasmids and sent them for sequencing at a bio company to ensure the correct sequence. The sequencing results confirmed that the plasmids were indeed the ones we wanted, with the correct sequence and no mutations.

![](https://static.igem.wiki/teams/5526/eng/24.jpg)

Figure 24. PCR identification of plactate1-Azurin plasmid.
A. The graph shows the length of p1-Azurin is 444bp.
The graph shows the flora growing in a petri dish.
C. The graph shows the sequencing measured by the bio company.

**Test 5:**

We used alkaline lysis to extract plasmids (plactate1-Azurin) from bacterial cultures. Next, we converted the recombinant plasmid to EcN1917 competent by heat shock transformation. PCR was used to verify whether the plasmid was transformed into EcN1917. The results are shown in Figure 25-A. The colony PCR results were detected by agarose gel electrophoresis, and the amplified band was 550bp, which was consistent with the target band, indicating that the four plasmids were successfully transformed into EcN1917.

![](https://static.igem.wiki/teams/5526/eng/25.jpg)

Figure 25. PCR identification of EcN1917 transformants.
A. The PCR production of the plactate1-Azurin transformants by agarose gel electrophoresis.
B. The graph shows the flora growing in a petri dish.

We grow the bacteria holding the plasmids under different OD values (0.3, 0.6, 0.8,1.0) and lactic acid concentrations (0mM, 2mM, 5mM, 10mM). We used the nanodrop to detect protein concentration and created a graph of four different bacteria’s protein expression. We conclude that the protein concentration is highest when OD600 equals 0.6, and the lactic acid concentration is 5mM. We used SDS-PAGE to ensure we got the proteins we wanted.

![](https://static.igem.wiki/teams/5526/eng/26.jpg)

Figure 26. The effects of bacterial concentration and lactic acid concentration on the expression of plactate1-Azurin.

**Learn 5:**

This plasmid was constructed to compare Plldr(new)-Azurin with Plldr-Azurin(plactate1-Azurin). If the concentration of proteins produced by the Plldr(new)-Azurin is higher than that produced by Plldr-Azurin(plactate1-Azurin), it can show that improving the new Plldr is useful.

Cycle 6：Plldr(new)-Azurin (BBa\_K5526007)

**Design 6:**

In the plasmid Plldr(new)-Azurin (BBa\_K5526007) we construct, we combined Plldr-new (BBa\_K5526001), Azurin (BBa\_K5526000), and pUC57-mini (BBa\_K3983004) together to form Plldr(new)-Azurin (plactate2- Azurin).

Plldr(new) gets several improvements to the original promoter. It is more accurate and will not get inhibited within low oxygen concentrations. Azurin is a gene that encodes a chemical drug, which can release some medicines and substances to kill the tumor cells. pUC57-mini is the skeleton of the plasmid. Plldr(new)-Azurin (plactate2- Azurin) is the plasmid that will be activated and produce a toxic protein by high lactic acid concentration. In addition, it will not be limited within low oxygen concentration, like what the original one did instead.

![](https://static.igem.wiki/teams/5526/eng/27.png)

Figure 27. The plasmid map Plldr(new)-Azurin

**Build 6:**

We applied PCR on the genes Azurin(444bp) and the new pUC57-plldr (3800bp). We used agarose gel electrophoresis to check the length of our DNA pieces to ensure we succeeded. The result shows that the new pUC57-plldr has a length of 3800bp, and the Azurin has a length of 444bp.

![](https://static.igem.wiki/teams/5526/eng/28.jpg)

Figure 28. The identification of PCR production by agarose gel electrophoresis
Left: The graph shows that plactate 2 has a length of 3800 bp.
Right: The graph shows that p2-Azurin has a length of 444bp.

We first used homologous recombination to combine Azurin with the new pUC57-plldr promoter, forming the Plldr(new)-Azurin (plactate 2-Azurin). We then performed a heat shock conversion to make DH5α cells sensitive to frequent changes in temperature, alternating between high and low temperatures to facilitate the uptake of plasmids of DH5α. After heat shock, we injected the plasmids into DH5α cells and grew them on an Amp+ medium, ensuring that only bacteria containing the plasmids would survive. As expected, bacterial colonies grew on the petri dishes, indicating successful plasmid uptake. To further confirm the presence of the desired plasmid, we performed a colony PCR directly from the colonies on the plate. This allowed us to amplify the specific region of the plasmid containing the Plldr(new)-Azurin (plactate 2-Azurin) construct. Figure 26 shows the PCR results were positive, indicating that the colonies contained the correct plasmid. Finally, we recycled the plasmids and sent them for sequencing at a bio company to ensure the correct sequence. The sequencing results confirmed that the plasmids were indeed the ones we wanted, with the correct sequence and no mutations.

![](https://static.igem.wiki/teams/5526/eng/29.jpg)

Figure 29. PCR identification of plactate2-Azurin plasmid.
A. The graph shows the length of p2-Azurin is 444bp.
B. The graph shows the flora growing in a petri dish.
C. The graph shows the sequencing measured by the bio company.

**Test 6:**

We used alkaline lysis to extract plasmids (plactate2-Azurin) from bacterial cultures. Next, we converted the recombinant plasmid to EcN1917 competent by heat shock transformation. PCR was used to verify whether the plasmid was transformed into EcN1917. The results are shown in Figure 30-A. The colony PCR results were detected by agarose gel electrophoresis, and the amplified band was 550bp, which was consistent with the target band, indicating that the four plasmids were successfully transformed into EcN1917.

![](https://static.igem.wiki/teams/5526/eng/30.jpg)

Figure 30. PCR identification of EcN1917 transformants.
A. The PCR production of the plactate2-Azurin transformants by agarose gel electrophoresis.
B. The graph shows the flora growing in a petri dish.

We grow the bacteria holding the plasmids under different OD values (0.3, 0.6, 0.8,1.0) and lactic acid concentrations (0mM, 2mM, 5mM, 10mM). We used the nanodrop to detect protein concentration and created a graph of four different bacteria’s protein expression. We conclude that the protein concentration is highest when OD600 equals 0.6, and the lactic acid concentration is 5mM. We used SDS-PAGE to ensure we got the proteins we wanted.

![](https://static.igem.wiki/teams/5526/eng/31.jpg)

Figure 31. The effects of bacterial concentration and lactic acid concentration on the expression of plactate 2-Azurin.

**Learn 6:**

This plasmid was constructed to compare Plldr(new)-Azurin (plactate 2-Azurin) with Plldr-Azurin. If the concentration of proteins produced by the Plldr(new)-Azurin (plactate 2-Azurin) is higher than that produced by Plldr-Azurin, it can show that improving the new Plldr is useful.

Other Test

After the optimal protein expression conditions were obtained, the constructed strains (plactate1-Azurin/Anti-PD-L1, plactate2-Azurin/Anti-PD-L1) were expanded for culture, and 5mM lactic acid was used to induce the actual expression level of tumor drugs in EcN1917. The experimental results are shown in Figure 32. The size of the Azurin protein is about 19kDa, and the Anti-PD-L1 protein is about 24kDa, consistent with the expected results. A large amount of target protein indicates that the Azurin/Anti-PD-L1 protein is successfully expressed. In the future, we will further study the expression and tumor inhibition effect of the constructed probiotics in the tumor environment.

![](https://static.igem.wiki/teams/5526/eng/32.jpg)

Figure 32 Detection of Azurin and Anti-PD-L1 protein expression by SDS-PAGE

Summary:

EcN drug molecular delivery carriers are characterised by good compliance, long-lasting efficacy, and therapeutic precision. However, many issues still need to be resolved before they can be placed on the market.

We will conduct animal experiments in the future to verify the tumor inhibition effect of our engineered probiotics in animals. Existing studies have shown that EcN strains may not be ideal in clinical trials, so we need more trials and studies to produce more effective and safer EcN strains that can be used as probiotic drugs. Therefore, there is still a long way to go to achieve precision treatment of tumors using EcN drug molecular delivery vectors.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
{% block page\_content %}

![](https://static.igem.wiki/teams/5526/desc-banner.png)

{{ self.title() }}

* Constructing the plasmid
* Plasmid Extraction
* Polymerase Chain Reaction (PCR)
* Agarose Gel Electrophoresis
* DNA Gel Extraction
* Homologous Recombination
* E.coli Transformation
* Colony PCR
* Protein Expressions
* Protein Gel Electrophoresis

Constructing the plasmid

**Preparing Luria - Bertani (LB) medium**

**Goal: Used to cultivate E. coli**

| Apparatus | Amount |
| --- | --- |
| Sterilised duran bottle, cup | 2 |
| Autoclave | 1 |
| Electronic Balance | 1 |

**Liquid Luria - Bertani a (Liquid LB) materials**

| Reagents | Volume/Mass |
| --- | --- |
| Tryptone | 10g |
| Yeast extract | 5g |
| Sodium chloride (NaCl) | 10g |
| Double Distilled water (ddH2O) | 1000 mL |

**Procedures:**

✓ To prepare 1 liter of liquid LB medium, add 10g of tryptone, 5g of yeast extract, and 10g of NaCl to
950 mL of deionized water.
✓ Stir the container until the solutes dissolve, and adjust the pH to 7.0 using 5 M NaOH. Fill up to 1
liter with deionized water.
✓ Sterilize by steam for 15 minutes at 121℃.
**Solid Luria - Bertani (Solid LB) materials**

| Reagent | Volume/Mass |
| --- | --- |
| Liquid LB | 100 mL |
| Agar | 1.5g |

 **Procedures:**
✓ First, add 1.5 g of agar powder to 100 mL of liquid LB medium to prepare the solid LB medium.
✓ Place the molten LB agar medium in a 55°C water bath after high-pressure sterilization.
✓ When the temperature drops to 55°C, add the antibiotics to prevent deactivation due to high
temperature, and mix thoroughly.
✓ Pour 10 mL of the medium onto each plate. After pouring the mixed medium into Petri dishes, open the
lids and expose them to UV light for 10-15 minutes, sealing the edges with sealing tape.

Plasmid Extraction

**Goal: Remove RNA to separate plasmid DNA from bacterial genomic DNA, then remove proteins and other
impurities to obtain relatively pure plasmid DNA.**

| Apparatus | Amount |
| --- | --- |
| Centrifuge | 1 |
| Spin column | 4 |
| Thermostat | 1 |

| Reagents | Volume/Mass |
| --- | --- |
| Solution I: Bacterial Suspension | 250 μL/time |
| Solution II: Cell Lysis Buffer | 250 μL/time |
| Solution III: Neutralization Buffer | 350 μL/time |
| Wash solution | 750 μL/time |

**Procedures:**
1. Centrifugation and Pellet Preparation
Centrifuge 1-5 mL of bacterial culture (P1, P2, Azurin, anti-PD-L1) at 12,000 rpm for 1 minute and discard the
supernatant.

2. Resuspension
Add 250 µL of Solution I to the pellet in the centrifuge tube and use a vortex mixer to resuspend the
bacterial pellet thoroughly.

3. Lysis
Add 250 µL of Solution II to the tube and gently invert 6-8 times to fully lyse the bacteria; the solution
should become clear and viscous.

4. Precipitation
Add 350 µL of Solution III to the tube, invert 6-8 times to mix thoroughly, and a white precipitate should
form.
Centrifuge at 12,000 rpm for 10 minutes and transfer the supernatant to a clean centrifuge tube.

5. Precipitation with Ethanol
Add 0.35 volumes of absolute ethanol to the supernatant and mix thoroughly.

6. Column Loading and Initial Centrifugation
Transfer the supernatant to a column, allow it to sit at room temperature for 2 minutes, centrifuge at 12,000
rpm for 1 minute, discard the waste in the collection tube, and place the column back into the collection
tube.

7. Wash Steps
Add 750 µL of wash buffer to the column, centrifuge at 12,000 rpm for 1 minute, and place the column back into
the collection tube.
Add 700 µL of wash buffer to the column, centrifuge at 12,000 rpm for 1 minute, and place the column back into
the collection tube.
Centrifuge at 12,000 rpm for 2 minutes, then place the column open side up at room temperature or in a 50°C
incubator for several minutes.

8. Elution
Place the column into a clean centrifuge tube, and add 50-200 µL of pre-warmed elution buffer (65°C) to the
center of the membrane; allow it to sit at room temperature for 2 minutes, then centrifuge at 12,000 rpm for 1
minute.
Repeat the elution by adding the collected elution buffer back to the column, allowing it to sit at room
temperature for 2 minutes, and centrifuge at 12,000 rpm for 1 minute.

Polymerase Chain Reaction (PCR)

**Goal： In vitro, DNA amplification technology can rapidly and specifically amplify any target DNA outside of
a living organism.**

| Apparatus | Amount |
| --- | --- |
| Pipette (range: 2-20μL) | some |
| Pipette (range: 20μL-100μL) | some |
| Centrifuge | 1 |
| PCR amplification instrument | 1 |

| Reagents | Volume/Mass |
| --- | --- |
| Target gene (Plactate1-Azurin; Plactate1-anti-PD-L1; Plactate2-Azurin; Plactate2-anti-PD-L1) | 2 μL |
| 2\*Mix (dNTPs, DNA polymerase, Buffer, Magnesium ions) | 25 μL |
| Primers (F/R) | F: 2 μL R: 2 μL |
| ddH2O | 19 μL |

**Procedures:**

✓ Add different target genes, mix solution, primers, and double distilled water into each tube, and
prepare the initial solutions.
✓ Setup the PCR program

| Progress | Temperature | Time |  |
| --- | --- | --- | --- |
| Initial Denaturation | 95 ℃ | 5 min |  |
| Denaturation | 95 ℃ | 30s | repeat 30 times |
| Annealing | 55 ℃ | 30s |
| Extension | 72 ℃ | 1kb/min |
| Final Extension | 72 ℃ | 10 min |  |
| Hold | 4 ℃ | ∞ |  |

Agarose Gel Electrophoresis

**Goal: Separation and Identification of DNA and RNA Molecular Mixtures**

| Apparatus | Amount |
| --- | --- |
| Pipette (range: 2μL-20μL) | some |
| Pipette (range: 20μL-200μL) | some |
| Weighing paper | 1 piece |
| Electronic balance | 1 |
| Microwave oven | 1 |
| Spatula | 1 |
| Graduated cylinder | 1 |
| Comb | 2 |
| Electrophoresis chamber | 1 |

| Reagents | Volume/Mass |
| --- | --- |
| Agarose | 5g |
| Tris Acetate-EDTA buffer (TAE) | 50 mL |
| Red dye | 3μL |
| Marker | 5μL |

**Procedures:**

1.Preparing the gel
✓ Place the weighing paper on the electronic balance and weigh 5g of agarose. Transfer it to a conical
flask.
✓ Add 50 mL of TAE buffer to the conical flask and mix.
✓ Microwave it for 3 minutes until the agarose granules dissolve completely.
✓ Use a pipette (2μL-20μL range) to add 5 μL of nucleic acid dye to the flask.
✓ Pour the mixture into the mold and insert a comb.
✓ Wait 10 minutes for the gel to solidify.
2.Electrophoresis
✓ Install the electrophoresis chamber.
✓ Pour the cooled gel gently onto the horizontal plate of the electrophoresis chamber.
✓ After it solidifies, add electrophoresis buffer into the chamber and remove the comb.
✓ Add the sample mixture to the wells.
✓ Connect one end of the electrophoresis wells to the negative electrode and the other end to the
positive electrode. Turn on the power, set the voltage to 180V, and run electrophoresis for 20 minutes.
✓ Remove the gel and observe under UV light.
✓ DNA bands will appear as red fluorescent bands.

DNA Gel Extraction

**Goal: Extract pure target gene bands and apply them in forward experiments**

| Apparatus | Amount |
| --- | --- |
| Scalpel | 1 |
| UV transilluminator | 1 |

| Reagents | Volume/Mass |
| --- | --- |
| Sol liquid | 300μL/0.1g DNA gel block |
| Rinse solution | 600μL/time |

**Procedures:**

1. After agar-gel electrophoresis, a single DNA strip of interest is cut from the agar-gel and weighted
2. Add 600μL sol solution to the gel block and place in a water bath at 55℃ for 10 min. During the process,
gently turn the centrifuge tube up and down continuously to ensure that the glue block is fully dissolved
3. The solution was added to an adsorption column and centrifuged at 12000pm for 60 seconds
4. Add 600μL bleaching solution to the adsorption column and centrifuge at 12000rpm for 1min, discard the
waste liquid, and put the adsorption column into the collection tube
5. Add 600μL bleach solution into the adsorption column, centrifuge 12000rpm for 1min, discard the waste
liquid, and put the adsorption column into the collection tube
6. Centrifuge at 12000rpm for 2min, remove the bleach solution as much as possible, and place the adsorption
column open at room temperature or 50℃ in a temperature box
7. Place the adsorption column into a clean centrifuge tube, drop the appropriate amount of eluent preheated
in a water bath at 65℃ into the center of the adsorption film, place at room temperature for 2min, and
centrifuge at 12000rpm for l min
8. DNA products stored at -20℃

Homologous Recombination

**Goal: Achieve the recombinant plasmids**

| Apparatus | Amount |
| --- | --- |
| Pipettes (range: 2-20μL/20-200μL) | 4 |
| Water bath | 1 |

| Reagents | Volume/Mass |
| --- | --- |
| Linearized plasmid | 2μL |
| Target gene | 1μL |
| 2 × ClonExpress Mix | 5μL |
| ddH2O | 2μL |

**Procedures:**

Prepare the following reaction system on the ice. Use a pipette to gently suck and mix (do not shake and mix), and briefly centrifuge to collect the reaction liquid to the bottom of the tube. 50℃, 15 min; Reduce to 4 ° C or cool immediately on ice

E.coli Transformation

**Procedures:**

1. Inoculating ECN 1917 and preparing competent ECN 1917 cells
Inoculate overnight ECN 1917 culture with 1% volume into fresh medium and grow at 37°C, 220 rpm until OD600 reaches 0.3 (2.5 hours).
Harvest the bacteria by chilling on ice for 5 minutes, then centrifuge at 4°C, 5000 rpm for 5 minutes.
Wash the pellet with cold deionised water and centrifuge at 4°C, 5000 rpm for 5 minutes.
Resuspend the pellet in 2 mL of cold 0.05 M CaCl2 and 15% glycerol solution, gently mix, and chill on ice for 5 minutes.
Add another 2 mL of cold 0.05 M CaCl2 and 15% glycerol solution and gently mix to obtain competent cell suspension. Aliquot into 50-100 µl portions and store at -70°C.

2. Heat recovery
Heat the sample at 42°C for 45 seconds, then immediately place it on ice to cool for 2-3 minutes.

3. Recovery
Add 900 μL of LB medium (without antibiotics) to each tube.
Gently mix the contents and incubate the tubes in a shaking incubator at 37°C and 200 rpm for 45-60 minutes to allow the cells to recover and express the plasmid-encoded genes.

4. Plating
Centrifuge at 5000 rpm (2,500 × g) for 5 min and discard 900 μL supernatant. The bacteria sediment was suspended with the remaining medium and lightly coated with a sterile coating stick on a plate containing Amp resistance.
Then, invert the plates and incubate at 37°C for 12-16 hours to allow colonies to grow.

Colony PCR

**PCR program**

| Apparatus | Amount |
| --- | --- |
| Pipette (range: 2-20μL) | 1 |
| Pipette (range: 20μL-100μL) | 1 |
| Centrifuge | 1 |
| PCR amplification instrument | 1 |

| Reagents | Volume/Mass |
| --- | --- |
| Target gene (Plactate1-Azurin; Plactate1-anti-PD-L1; Plactate2-Azurin; Plactate2-anti-PD-L1; DNA polymerase; Magnesium ions) | 2μL |
| 2\*Mix (dNTPs, DNA polymerase, Buffer, Magnesium ions) | 10μL |
| Primers (F/R) | F: 1μL R: 1μL |
| ddH2O | 8μL |

**Procedures:**

✓ Add different target genes, mix solution, primers, and double distilled water into each tube, and prepare the initial solutions.
✓ Setup the PCR program

| Progress | Temperature | Time |
| --- | --- | --- |
| Initial Denaturation | 95 ℃ | 5 min |
| Denaturation | 95 ℃ | 30s |
| Annealing | 55 ℃ | 30s (repeat 30 times) |
| Extension | 72 ℃ | 1 min |
| Final Extension | 72 ℃ | 10 min |
| Hold | 4 ℃ | ∞ |

**Gel Electrophoresis**

Prepare the Agarose Gel:
✓ Gel Preparation: Dissolve agarose powder in a TAE buffer solution by heating. The agarose concentration typically ranges from 0.7% to 2.0%, depending on the size of the DNA fragments you want to resolve.
✓ Cooling and Casting: Allow the agarose solution to cool to about 50-60°C before pouring it into a gel casting tray. Insert a comb to create wells for loading the samples. Let the gel solidify at room temperature (30 minutes).
Prepare the Gel Electrophoresis Apparatus:
✓ Setup: Place the solidified agarose gel into the electrophoresis tank and cover it with a running buffer (TAE).
✓ Buffer Preparation: Prepare the electrophoresis buffer according to the manufacturer's instructions. This buffer facilitates the conduction of electricity through the gel and helps maintain the pH.
Load the Samples:
✓ Mix with Loading Dye: Mix the PCR products with a loading dye. This dye helps to visualize the sample and adds density so that the samples sink into the wells.
✓ Loading: Carefully pipette the PCR products and a DNA ladder into the gel wells.
Run the Gel:
✓ Electrophoresis: Apply an electric current across the gel. DNA fragments will migrate through the gel matrix towards the positive electrode (anode) because DNA is negatively charged. Smaller fragments move faster and travel further than larger ones.
Visualize the DNA:
✓ Staining: After electrophoresis, stain the gel with a DNA-binding dye. This dye intercalates with the DNA and fluoresces under UV light.
✓ Imaging: Use a gel documentation system or UV transilluminator to visualize and capture an image of the gel. Bands corresponding to DNA fragments will appear, allowing you to compare the size of the PCR products with the DNA ladder.

Protein Expressions

**GFP Fluorescence Measurement**

✓ Fluorescence Microscopy Detection: sfGFP fluorescence emission.
✓ Spectrophotometer Measurement:
Black 96-well plates, 470 nm excitation, 509 nm emission, and read the value;
Transparent 96-well plates, OD600;
Relative fluorescence intensity = read value of the former / read value of the latter.

Protein Gel Electrophoresis

| Apparatus | Amounts |
| --- | --- |
| Gel Molds | 2 |
| Pipettes and pipette tips (2-20μL / 100-1000μL) | 2 |
| Magnetic Stirrer | 1 |
| Stirring Rod | 1 |
| Water bath machine | 1 |

| Reagents | Volume or Mass |
| --- | --- |
| Tris-HCl (1.5 M, pH 8.8) | 2.5 mL (for a 12% gel) |
| Tris-HCl (0.5 M, pH 6.8) | 1.25 mL (for a 12% gel) |
| ddH2O | Up to the top of the gel mold |
| Sample Buffer (different lactic acid concentrations of P1, P2, anti-PD L1, Azurin) | 10 µL per sample |
| Electrophoresis Buffer | 1 L (Tris-Glycine-SDS buffer) |
| Coomassie Brilliant Blue | 5 µL |

**Procedures:**

1. Prepare Equipment and Materials
✓ Gel electrophoresis equipment (electrophoresis tank, power supply)
✓ Gel components: acrylamide, crosslinker (N, N'-methylene bisacrylamide), buffer solution
✓ Sample buffer (P1, P2, Anti-PD L1, Azurin)
✓ Coomassie Brilliant Blue

2. Gel Preparation
2.1 Prepare Separating Gel
✓ Mix acrylamide solution: acrylamide, crosslinker, and buffer solution.
✓ Pour into gel mold: Usually, the separating gel is poured first and then allowed to solidify.
2.2 Prepare Stacking Gel
✓ Mix stacking gel solution: acrylamide, crosslinker, and buffer solution.
✓ Pour into gel mold on top of the solidified separating gel.
2.3Insert Comb
✓ Insert a comb to form wells in the gel, ensuring it solidifies with the wells intact.
2.4 Solidify Gel
✓ Allow the gel solution in the mold to solidify at room temperature or in a 4°C refrigerator, typically for 30 minutes to 1 hour.

3. Sample Preparation
3.1 Process Samples
✓ Mix samples with sample buffer, usually containing SDS and reducing agents.
✓ Heat samples (usually at 95°C for 5-10 minutes) to denature and linearize proteins.
3.2 Cool Samples
✓ Cool the samples to room temperature after heating and prepare for loading.

4. Electrophoresis Steps
4.1 Assemble Gel
✓ Remove the solidified gel from the mold and place it in the electrophoresis tank.
✓ Add electrophoresis buffer to ensure it covers the gel.
4.2 Load Samples
✓ Carefully load the prepared samples into the gel wells using a micropipette.
4.3 Run Electrophoresis
✓ Connect the electrophoresis tank to a power supply and set the appropriate voltage (120V).
✓ Run electrophoresis until the dye front reaches the bottom of the gel (usually 1 hour).

5. Post-Processing
5.1 Stain Gel
✓ Treat the gel with a staining reagent (Coomassie Brilliant Blue) to visualize protein bands.
5.2 Destain Gel
✓ After staining, place the gel in a destaining solution or water to remove background staining and reveal clear protein bands.
5.3 Analyze Results
✓ Observe and record the position and intensity of protein bands.
✓ Estimate protein molecular weights using molecular weight markers.

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![](https://static.igem.wiki/teams/5526/desc-banner.png)

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* Overview
* Plasmid Construction
* EcN1917 Transformation
* Protein Expression and Identification
* Future plan
* References

Overview

According to the World Health Organization, cancer is a leading cause of death worldwide, causing nearly 10 million (or almost one in six) deaths in 2021. Genetically engineered bacteria are gaining increasing attention as a novel disease diagnosis and treatment strategy. Engineered bacteria have been used to monitor liver metastases, liver dysfunction, and gastrointestinal diseases2. In addition, a variety of bacteria, such as ECN (Nissle1917, probiotics), have been shown to selectively colonize tumors because they can infiltrate tumors, especially the hypoxic core of immune deficiency. As a probiotic injection drug, it takes advantage of the tumor targeting of bacteria, tracks the tumor, enriches it, and releases the protein drug. Based on synthetic biology, we designed a precise probiotic therapy. After the probiotics were colonized into the tumors, they recognized the lactic acid environment around them, and the lactic acid-responsive promoter activated the expression of tumor drugs, accurately controlled the bacterial activity in tumor tissues and the expression and release of drugs, and further improved the safety and effectiveness of probiotic cancer therapy.

![](https://static.igem.wiki/teams/5526/result/1.jpg)

Plasmid Construction

To complete plasmid construction, we connected the skeleton (pUC57), promoter (pLldR), and target gene. We used two types of promoters: pLldR (referred to as plactate1) and pLldR-New (referred to as plactate2). The Lactate1 promoter is a lactate-responsive promoter that strongly initiates tumor drug expression in a lactate environment. This promoter is often used as a lactic acid biosensor for specific gene construction. Ana Zuniga's work shows that the plactate2 promoter is an optimized, synthetic lactate biosensor system designed to incorporate hybrid promoters of pLldR operators, remove glucose breakdown metabolites and hypoxia inhibition, and adjust the expression of regulatory genes and reporter genes. Plactate2 can still activate the expression of tumor drugs in the low oxygen environment3. Therefore, we attached plactate1 and plactate2, respectively, to three different target genes (sfGFP, Azurin, and Anti-PDL1) to screen for the optimal lactic acid promoter pLldR and transferred the constructed recombinant plasmid to E. coli Nissle1917. The follow-up will provide a new idea for tumor treatment.

**1.1plactate1-sfGFP, plactate2-sfGFP plasmid construction**

The target gene sfGFP was obtained by PCR. sfGFP is a fluorescent protein, and the addition of sfGFP allows us to detect the success of plasmid construction. At the same time, the ability of the two promoters to initiate expression can be further compared. We obtained promoter (plactate1 and plactate2) and sfGFP fragment of the target gene by PCR amplification. Then, we detected the DNA length by gel electrophoresis and obtained that the length of plactate1 homologous arm-sfGFP and plactate2 homologous arm-sfGFP were both 750bp (as shown in Figure 1).

![](https://static.igem.wiki/teams/5526/result/2.jpg)

Figure 1. The identification of PCR production by agarose gel electrophoresis
Left: the graph shows plactate 1 and plactate 2 has the length of 3150bp and 3800bp.
Right: the graph shows that p1-sfGFP and p2-sfGFP have a length of 750bp.

Then, through homologous recombination, we connected the target gene sfGFP and promoter (plactate1 and plactate2) to the skeleton to obtain Plactate1-SFGFP, Plactate2-sfGFP and then verified the successful construction of the recombinant plasmid by colony PCR. As can be seen from Figure 2-A, the amplified band was 750bp, which was consistent with the expected result, indicating that the initial construction of the recombinant plasmid was successful. Subsequently, we extracted the constructed plasmid and sent it to a biological company for sequencing. The sequencing results were compared with the sfGFP sequence one by one. As shown in Figure 2-C, the gene sequences of the two plasmons were matched entirely, which further proved that the recombinant plasmid was successfully constructed.

![](https://static.igem.wiki/teams/5526/result/3.jpg)

Figure 2. PCR identification of plactate1-sfGFP plasmid.
A. The graph shows that the lengths of p1-sfGFP and p1-sfGFP are both 750 bp. B. The graph shows the flora growing in petri dish. C. The graph shows the sequencing measured by the bio company.

**1.2plactate1-Azurin, plactate2-Azurin, plactate1-Anti-PD-L1 and plactate2-Anti-PD-L1 plasmid construction**

According to Ana Zuniga's article, Azurin, a cuproredoxin isolated from Pseudomonas aeruginosa, can enter cancer cells and induce apoptosis. It interacts with specific molecules on the surface of cancer cells to disrupt intracellular signaling pathways. The pLldR promoter, derived from Lactobacillus plantarum, is a lactate-induced promoter known for its ability to respond to lactic acid. It is a moderately strong promoter that controls the expression level of target genes. We attached Azurin and Anti-PD-L1 to the skeleton (pUC57) with plactate1 and plactate2, respectively. We obtained promoters (plactate1 and plactate2) and target genes (Azurin and Anti-PD-L1) by PCR amplification. Next, we measured the length of the PCR products by gel electrophoresis. The lengths of plactate1 homologous arm-Azurin, plactate2 homologous arm-Azurin, plactate1 homologous arm-Anti-PD-L1, and plactate2 homologous arm-Anti-PD-L1 are all 750bp (as shown in Figure 3-A).

![](https://static.igem.wiki/teams/5526/result/4.jpg)

Figure 3 The identification of PCR production of plasmid and Azurin/Anti-PD-L1gene.
A. PCR production by agarose gel electrophoresis
B. The proof diagram of PCR

Then, through homologous recombination, we attach target genes (Azurin and Anti-PD-L1) to the skeleton, plactate1-Azurin, plactate2-Azurin, plactate1-Anti-PD-L1, and plactate2-Anti-PD-L1 were obtained, and then colony PCR was used to verify whether the recombinant plasmid was successfully constructed. As can be seen from Figure 4-A, the amplified bands were 750bp, which was consistent with the expected result, indicating that the initial construction of the recombinant plasmid was successful. Subsequently, we extracted the constructed plasmid and sent it to a biological company for sequencing. The sequencing results were compared with the sequences of the target genes (Azurin and Anti-PD-L1) one by one. As shown in Figure 4-C, the gene sequences of the two plasmids were matched entirely, which further proved that the recombinant plasmid was successfully constructed.

![](https://static.igem.wiki/teams/5526/result/5.jpg)

Figure 4 PCR identification of the recombinant plasmid plactate1-Azurin, plactate2-Azurin plactate1-Anti-PD-L1, and plactate2-Anti-PD-L1
A. The PCR production identification by agarose gel electrophoresis.
B. The graph shows the flora growing in petri dish.
C. The graph shows the sequencing measured by the bio company.

EcN1917 Transformation

We used alkaline lysis to extract plasmids (plactate1-Azurin, plactate1-Anti-PD-L1, plactate2-Azurin, plactate2-Anti-PD-L1) from bacterial cultures. Next, we converted the recombinant plasmid to EcN1917 competent by heat shock transformation. PCR was used to verify whether the plasmid was transformed into EcN1917. The results are shown in Figure 5-A. The colony PCR results were detected by agarose gel electrophoresis, and the amplified band was 550bp, which was consistent with the target band, indicating that the four plasmids were successfully transformed into EcN1917.

![](https://static.igem.wiki/teams/5526/result/6.jpg)

Figure 5 PCR identification of EcN1917 transformants
A. The PCR production of the plactate1-Azurin, plactate2-Azurin, plactate1-Anti-PD-L1, and plactate2-Anti-PD-L1 transformants by agarose gel electrophoresis.
B. The graph shows the flora growing in petri dish.

Protein Expression and Identification

We formulated different lactic acid concentrations (0mM, 2mM, 5mM, 10mM) to simulate different tumor microenvironments. The optimal promoter was selected by comparing the fluorescence protein intensity and the expression concentration of tumor drugs, which provided a new idea for future tumor treatment.

**3.1 The induced expression of fluorescent protein sfGFP**

The constructed strains (plactate1-sfGFP, plactate2-sfGFP) were expanded in culture, and different concentrations of lactic acid (0mM,2mM,5mM,10mM) were used to induce the expression of GFP fluorescent protein, as shown in Figure 6. It can be seen from Figure 6 that when the lactate concentration is 5mM, the fluorescence protein expression of the two strains is the strongest. Through horizontal comparison, the expression of the fluorescent protein of Plactate2-GFP is more vital than that of Plactate1-GFP, and it can be concluded that the plactate2 promoter has a stronger activation expression ability than the plactate1 promoter.

![](https://static.igem.wiki/teams/5526/result/7.jpg)

Figure 6 The effects of lactic acid concentration on the expression of fluorescence protein

**3.2 Determine the optimal culture OD and lactic acid induced concentration**

In order to further determine the optimal conditions for the growth of bacterial solution, we set up different bacterial solution cultures of OD600 (OD0.3, OD0.6, OD0.8, and OD1.0) and different lactate induction concentrations (0mM, 2mM, 5mM, and 10mM). Four strains were cultured to OD0.3, OD0.6, OD0.8, and OD1.0, and different concentrations of lactic acid were added for induction. After induction, the two target proteins were extracted by ultrasonic crushing, and the concentration of target proteins was detected. As shown in Figure 7, when OD600 is 0.6 and lactic acid induced concentration is 5mM, the protein expression level is the highest. Therefore, the optimal conditions for protein expression can be determined as OD600=0.6 and lactic acid concentration is 5mM.

![](https://static.igem.wiki/teams/5526/result/8.jpg)

Figure 7 The effects of bacterial concentration and lactic acid concentration on the expression of recombinant proteins

Further, we processed the data using Origin Pro to form a three-dimensional mapping of the expressed protein concentrations of the four EcN1917 strains under different conditions (Fig. 8). By analyzing the 3D mapping, we can conclude that as far as the culture OD600 of the bacteriophage is concerned, there is a significant difference in the concentration of target proteins when induction is performed at different bacteriophage OD600. As can be seen from Figure 8, the highest level of protein expression was observed when the OD600 was 0.6. As for the lactic acid induced concentration, it is not the case that the higher the induced concentration, the highest the concentration of the target protein. As can be seen from Figure 8, when the final concentration of lactic acid was added to induce the expression, the highest level of the target protein was expressed.

![](https://static.igem.wiki/teams/5526/result/9.jpg)

Figure 8 3D mapping of the expressed protein concentrations

We further examined the change in the pH value of bacterial solution after different concentrations of lactic acid induced culture. The bacterial solution cultured overnight at 37 degrees Celsius was tested with a pH meter. When the lactic acid concentration is 0mM, the pH value of the bacterial solution is 6.87. When the lactic acid concentration is 2mM, the pH value of the bacterial solution is 6.60. When the lactic acid concentration is 5mM, the pH value of the bacterial solution is 6.42. When the lactic acid concentration is 10mM, the pH value of the bacterial solution is 6.08. When a tumor develops, the pH of the tumor microenvironment is about 6.5 to 7.2. When the pH value is less than 6.5, it will inhibit the killing effect of immune cells, thereby increasing the survival rate of tumor cells. When the pH value exceeds 7.2, it will affect the metabolism of tumor cells, membrane permeability, and other physiological processes, thus inhibiting its growth and spread. Therefore, when probiotics infiltrate tumors in a high lactic acid and low pH environment, tumor drugs are released to achieve the purpose of killing cancer cells.

![](https://static.igem.wiki/teams/5526/result/10.jpg)

Table 1 pH value of bacterial solution after different concentrations of lactic acid

**3.3 The expression of Azurin and Anti-PDL1 protein was induced**

After the optimal protein expression conditions were obtained, the constructed strains (plactate1-Azurin/Anti-PD-L1, plactate2-Azurin/Anti-PD-L1) were expanded for culture, and 5mM lactic acid was used to induce the actual expression level of tumor drugs in EcN1917. The experimental results are shown in Figure 8. The size of the Azurin protein is about 19kDa, and the Anti-PD-L1 protein is about 24kDa, consistent with the expected results. A large amount of target protein indicates that the Azurin/Anti-PD-L1 protein is successfully expressed. In the future, we will further study the expression and tumor inhibition effect of the constructed probiotics in the tumor environment.

![](https://static.igem.wiki/teams/5526/result/11.jpg)

Figure 9 Detection of Azurin and Anti-PD-L1 protein expression by SDS-PAGE

Future plan

ECN drug molecular delivery carriers are characterised by good compliance, long-lasting efficacy, and therapeutic precision. However, many issues still need to be resolved before they can be placed on the market.

Firstly, some engineered ECN strains that are effective in animal experiments could be more satisfactory in clinical trials, such as SYNB1020, an ECN strain tested in clinical trials4. Secondly, the stability and safety of recombinant strains in industrial production is also a concern because bacterial plasmids are genetically unstable and antibiotic genes in the plasmid are easily transferred. However, some of these problems can be solved by cloning foreign genes into the ECN genome5. In addition, some animal experiments are needed to determine whether the insertion of foreign genes affects bacterial metabolism, growth, reproduction, and the effect on the host5.

We will conduct animal experiments in the future to verify the tumor inhibition effect of our engineered probiotics in animals. Existing studies have shown that EcN strains may not be ideal in clinical trials, so we need more trials and studies to produce more effective and safer EcN strains that can be used as probiotic drugs. Therefore, there is still a long way to go to achieve precision treatment of tumors using ECN drug molecular delivery vectors.

References

[1]Cao, W., Chen, H.D., Yu, Y.W., Li, N. & Chen, W.Q. Changing profiles of cancer burden worldwide and in China: a secondary analysis of the global cancer statistics 2020. Chin Med J (Engl) 134, 783-791 (2021).

[2]Riglar, D.T. & Silver, P.A. Engineering bacteria for diagnostic and therapeutic applications. Nat Rev Microbiol 16, 214-225 (2018).

[3]Zuniga, A., et al. Engineered l-Lactate Responding Promoter System Operating in Glucose-Rich and Anoxic Environments. ACS Synth Biol 10, 3527-3536 (2021).

[4]R. Ochoa-Sanchez, M.M. Oliveira, M. Tremblay, G. Petrazzo, A. Pant, C.R. Bosoi, M. Perreault, W. Querbes, C.B. Kurtz, C.F. Rose, Genetically engineered E. coli Nissle attenuates hyperammonemia and prevents memory impairment in bile-duct ligated rats, Liver Int. 41 (2021) 1020–1032.

[5]Chen, H. , Lei, P. , Ji, H. , Yang, Q. , Peng, B. , & Ma, J. , et al. (2023). Advances in Escherichia coli nissle 1917 as a customizable drug delivery system for disease treatment and diagnosis strategies. Materials Today Bio, 18.

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