iGEM 2023 — Team nanjing-china

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- [Abstract](#s0)
- [Circle 1](#s1)
  * [Target 1: Obtain indigoidine dye](#s11)
  * [Target 2: Co-culture](#s12)
- [Circle 2](#s2)
  * [Target 3: Modifying *K. xylinus*](#s21)
- [References](#s3)

Indigoidine is a common jeans dye. The process of industrial manufacturing and jeans dyeing can cause serious problems such as water pollution and air pollution. In order to alleviate the environmental pollution caused by textile, Nanjing-China 2023 tries to use microbial cultivation technology to produce bacterial cellulose and dyes at the same time, and make the two into new colored textile fibers to replace traditional textile materials. Since *K. xylinus* can produce cellulose itself, we plan to obtain colored bacterial cellulose directly by cultivating modified *K. xylinus*. The first pigment our team has chosen to try is indigoidine, and if the experiment is successful and we have enough time, we will expand the pigment library and build a color fiber system. Making *K. xylinus* a cell factory for colored fibers provides a new paradigm for the textile industry.

# Circle 1

## Target 1: Obtain indigoidine dye ****Why indigoidine:**** Among the natural pigments, indigoidine is a natural blue pigment encoded by a single module-type nonribosomal peptide synthetase (Panchanawaporn et al.2022). Also, indigoidine has a simple biosynthetic pathway, involving the condensation of two moles of L-glutamine, and is amenable to microbial overproduction. So we chose indigoidine as the first dye for the experiment. ****Pre-experiment:**** , LINKS\_China 2021 produced indigo dye and Tharian purple dye from *Escherichia coli* and used these two dyes to stain BCM. Through their results, we found that the coloring effect of indigoidine on BCM is not very good, which may be related to the fat-soluble properties of these dyes. To verify this idea, we used the water-soluble dyes lemon yellow, carmine (these dyes have also been used by the igem team before, and we verify here to ensure the accuracy and repeatability of the experiment) and the liposoluble dye indigoidine to stain BCM, and the results are as follows. It can be seen from the results that the water-soluble dyes dye clearly and evenly. In contrast, the effect of indigoidine (DMSO extracting) dyeing is not very good, uneven and the color is lighter. Therefore, after reviewing the literature and brainstorming, we decided to obtain the target product by co-culture method, that is, to ****culture indigoidine-expressing and BCM-expressing bacteria together,**** in order to make the fat-soluble dye dyeing effect better. ![beaker](https://static.igem.wiki/teams/4605/wiki/engineering/engineering-1.png) Figure1. carmine (left), lemon yellow (middle) and indigoidine (right) staining result ****Design:**** At the beginning of the project, we plan to achieve the target product through co-cultivation. First we need to get indigoidine dye. Indigoidine synthase catalyzes the condensation of two L-glutamine molecules to form indigoidine. 4′-phosphingolinetransferase (PPTase) is required to activate indigoidine synthase, which is excited by introducing coenzyme A into the peptide carrier domain (PCP) of indigoidine synthase(Hui et al.2021). Therefore, co-expression of indigoidine synthase and PPTase is often required for indigoidine biosynthesis. As the pigment is biosynthesized via condensation of two L-glutamine residues, *Corynebacterium glutamicum (C. glutamicum)* was considered as an ideal production host because it carries strong fluxes for the biosynthesis of L-glutamate, a precursor of L-glutamine. *C. glutamicum* can also heterologously express indigoidine synthase and has the native pcpS gene (which expresses PPTase), which is a good carrier for synthetic dyes (Mohammad R et al.2021). ![beaker](https://static.igem.wiki/teams/4605/wiki/engineering/engineering-2.png) Figure2. Activation of bpsA and metabolic pathway of indigoidine ****Build:**** On the basis of Nanjing-NFLS, we constructed a plasmid system containing bpsA. *C. glutamicum* can change its metabolism and synthesize indigoidine by expressing this enzyme. This is the most basic part, and we have also optimized the codon of bpsA gene to increase indigoidine production. ****We will use pEKEX2 plasmid backbone, ligated with bpsA sequences to try to express bpsA in *C. glutamicum*.**** In order to control the start and stop of dye synthesis, we added lactose operons in the design of parts, so that the dye synthesis can be induced by lactose. So, we built BBa\_K437001, BBa\_K4376002 parts and verified the Nanjing\_NFLS’s parts BBa\_K4376003. ![beaker](https://static.igem.wiki/teams/4605/wiki/engineering/engineering-3.png) Figure3. Plasmid fragment of bpsA and the expression in *C. glutamicum* ****Test:**** After cultivating the modified *C. glutamicum*, we obtained the dye that meets the experimental requirements. We then ****measure the absorbance of the product dye, confirm that the product we have is indigoidine****, and characterize the yield. ![beaker](https://static.igem.wiki/teams/4605/wiki/engineering/engineering-4.jpg) Figure 4. Absorbance measurement of dye ****Learn:**** Through consulting the information, we have obtained many methods for fixing color and improving the stability of indigoidine. This provides a good knowledge base for our follow-up experiments. Target 2: Co-culture ****Pre-design:**** Because *K. xylinus* can produce cellulose itself, it is a relatively intuitive and groundbreaking idea to culture *K. xylinus* with *C. glutamicum.* At the beginning, our idea was to create a culture environment suitable for the growth of both bacteria, directly co-culture, so that it can be naturally colored. However, because indigoidine is fat-soluble and denser than water, it is secreted and sinks to the bottom of the test tube. *K. xylinus* are aerobic bacteria that produce cellulose membranes on the surface of culture medium. In this case, insufficient contact between the two will lead to poor dyeing effect. But shaking cultivation would prevent the cellulose from forming a film, so we came up with the next idea. **Pre-test:** Our second model requires a complete cellulose membrane. After conducting extensive literature research and discussions, we learned that bacterial culture medium is crucial for cultivating bacteria and increasing BCM production. ****Therefore, we performed numerous experiments to adjust the concentrations of glucose and ethanol to determine the optimal cultivation conditions.**** We collaborated with the team members in the dry lab to establish a model that simulated the interaction between glucose and ethanol, and determined ****the best cultivation conditions: glucose concentration ranging from 30g/L to 35g/L and ethanol concentration ranging from 0% to 0.5%.**** ![beaker](https://static.igem.wiki/teams/4605/wiki/engineering/engineering-5.png) Figure5. Optimal cultivation conditions ****Improved-design: We intend to utilize BCM as a framework to construct a growth scaffold for *C. glutamicum***** (Gilbert et al.2021). Essentially, we will first culture *K. xylinus* to produce BCM, and then introduce *C. glutamicum* to the existing BCM. In the culture device designed by us, the components added from top to bottom are inoculated *C. glutamicum*, BCM, and *C. glutamicum* medium. This approach effectively addresses the issue of dye contact with BCM and significantly enhances the likelihood of successful coloring. Moreover, it holds immense potential for future production and application. For instance, we can employ advanced techniques to design BCM in various shapes, making it easier to achieve products with diverse colors using our method. This approach offers flexibility to cater to the needs of the masses and pushes the boundaries of synthetic biology to new heights. During the co-cultivation process, the rotation speed of the shaker played a crucial role. If the rotation speed was too high, the cellulose would turn granular and fail to form a film. Conversely, if the rotation speed was too low, the dyeing could be uneven. ![beaker](https://static.igem.wiki/teams/4605/wiki/engineering/engineering-6.png) Figure6. Co-culture model ****Test:**** Practice our ideas. The *C. glutamicum* was inoculated on BCM and the staining results were observed. After dyeing for a period of time, we washed the BCM and observed the surface structure of the film under electron microscope. ****The mechanism of pigment coloring was studied by electron microscopy.**** ![beaker](https://static.igem.wiki/teams/4605/wiki/engineering/engineering-7a.jpg) Figure7a. Electron microscope diagram In addition, **we measured the growth curve of *K. xylinus*** to understand the growth status of the bacteria and lay a good foundation for subsequent experiments. ![beaker](https://static.igem.wiki/teams/4605/wiki/engineering/engineering-7b.png) Figure7b. Growth curve of K. xylinus **Learn:** Through this experiment, we found that the experimental results were affected by a variety of environmental factors, and it was difficult to meet the culture conditions of the two bacteria at the same time. The final staining results did also not match our expectations. So through discussion and exploration, we came up with a novel idea. **That is, the bpsA gene is induced to express in *K. xylinus*, so that it directly produces BCM containing dye.** **Cycle 1. Co-cultures** ![beaker](https://static.igem.wiki/teams/4605/wiki/engineering/engineering-cycle1.png) Up to now, we have completed the first cycle. Circle 2 Target 3: Modifying *K. xylinus* ****Design:**** There were some shortcomings in the co-culture experiment, so we decided to introduce the indigoidine synthesis plasmid into *K. xylinus.* **The strain itself can synthesize glutamine, so we only need to introduce bpsA and pcpS.** *K. xylinus* synthesizes glutamine by utilizing nitrogen and carbon sources in the medium. Glutamine can synthesize indigoidine under the action of bpsA activated by PPTase. This approach reduces bacterial usage, resulting in cost savings and promoting environmentally friendly practices. Moreover, only the growth conditions of single species of bacteria need to be adjusted, and the operation is simpler. Most importantly, this is the first time that dye plasmids have been introduced into *K. xylinus*, which is a very innovative and challenging idea. ****Build:**** We constructed two plasmids. ****EGFP(YFP) gene and bpsA-pcpS gene fragments were independently connected to plasmids**** by homologous recombination. The former was designed to verify whether the plasmid was electrocuted into *K. xylinus* allowing the results to be visualized. At the same time, we can also use it to see how cellulose wraps up *K. xylinus.* Most importantly, EGFP can be used to characterize promoter strength. When the plasmid with gene EGFP(YFP) is successfully expressed, we then electrically transfer the plasmid containing bpsA-pcpS into bacteria to produce our target product. **We will use a *K. xylinus* compatible PSB1A2 plasmid backbone, ligated with promoters such as strong promoters (J23100, J23104, etc.), and bpsA-pcpS sequences to try to express bpsA and PPTase in *K. xylinus*** (Hui et al.2021). ![beaker](https://static.igem.wiki/teams/4605/wiki/engineering/engineering-8.png) Figure8. Plasmid fragment of bpsA-pcpS and how color fiber are produced in *K. xylinus* ****Test:**** Detect fluorescence expression intensity and product expression. Due to time, we only **tested whether the plasmid was successfully constructed and introduced.** **Improve and rebuild:** The first results showed that the plasmid we inserted was not well expressed. After discussion, we reconstructed a plasmid and changed the promoter on the basis of the original plasmid to improve the intensity of gene expression. ****After reviewing the literature, we selected J23100/J23104/J23119 as the promoter of this experiment. This promoter has been verified to be reliable by many teams.**** **Further exploration:** If the project is successful, we can get different colors of cellulose by introducing different dye plasmids into *K. xylinus* . Thus, the application range of BCM can be expanded. In addition, we can also use the dyes produced by the previous team, such as spiruloviolet produced by BJU\_China 2012, curcumin and lycopene produced by HUST\_China 2021, and so on. The genes of pigments they use can be directly loaded into the plasmid we constructed to implement “Rainboweaver”, creating the color fiber system. **Cycle 2. Modifying *K. xylinus*** ![beaker](https://static.igem.wiki/teams/4605/wiki/engineering/engineering-cycle2.png) Finally, we completed the cycle of modification of *K. xylinus*. We believe that this transformation will play an important role in the BCM industry, and if we can successfully express colored cellulose, it will greatly reduce the cost of time and money, and also alleviate environmental pollution. We hope that this project can continue to develop and we will continue to study it. Reference [1] Panchanawaporn S, Chutrakul C, Jeennor S, Anantayanon J, Rattanaphan N, Laoteng K. Potential of Aspergillus oryzae as a biosynthetic platform for indigoidineidine, a non-ribosomal peptide pigment with antioxidant activity. PLoS One. 2022 Jun 23;17(6):e0270359. doi: 10.1371/journal.pone.0270359. PMID: 35737654; PMCID: PMC9223385. [2] Hui, Cy., Guo, Y., Li, Lm. et al. indigoidineidine biosynthesis triggered by the heavy metal-responsive transcription regulator: a visual whole-cell biosensor. Appl Microbiol Biotechnol 105, 6087–6102 (2021). [3] High-Level Production of the Natural Blue Pigment indigoidineidine from Metabolically Engineered Corynebacterium glutamicum for Sustainable Fabric Dyes. [4] Gilbert C, Tang TC, Ott W, Dorr BA, Shaw WM, Sun GL, Lu TK, Ellis T. Living materials with programmable functionalities grown from engineered microbial co-cultures. Nat Mater. 2021 May;20(5):691-700. doi: 10.1038/s41563-020-00857-5. Epub 2021 Jan 11. PMID: 33432140.

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- [Double enzyme digestion](#s1)
- [PCR for inserts](#s2)
- [Recombination](#s3)
- [Transformation](#s4)
- [Colony PCR](#s5)
- [Agarose Gel Electrophoresis](#s6)
- [Extract the plasmid](#s7)
- [Preparation of Competent Komagataeibacter xylinus](#s8)
- [Electroporation](#s9)
- [Preparation of HS culture medium](#s10)
- [Measurement of the K. xylinus growth curve](#s11)

# Double enzyme digestion

### Materials

Spe I
EcoR I
pSB1A2
10×Buffer
ddH2O

### Protocol

1. Prepare the reaction solution according to the following table：

   ---

   EcoR I

   1 µl

   Spe I

   1 μl

   pSB1A2

   2 µg

   10×Buffer

   5 µl

   ddH2O

   to 50 µl

   ---
2. 37 ℃ water bath insulation for 50 minutes

# PCR for inserts

### Materials

Template
2×Phanta
Primer F
Primer R
ddH2O

### Protocol

1. Prepare the reaction solution according to the following table：

   ---

   Template

   10 ng

   2×Phanta

   25.0 µl

   Primer F

   2.0 µl

   Primer R

   2.0 µl

   ddH2O

   to 50.0 µl

   ---
2. Run the PCR reaction：

   ![beaker](https://static.igem.wiki/teams/4605/wiki/protocol2-1.png)

# Recombination

Vazyme | ClonExpress MultiS One Step Cloning Kit（C113）

### Materials

Linearized vector
Insert
5 × CE MultiS Buffer
Exnase MultiS
ddH2O

### Protocol

1. Dilute the vector and insert fragments at an appropriate ratio before preparing the recombination reaction system, and the amount of each component is not less than 1 µl.
2. Prepare the following reaction on ice:

   ![](https://static.igem.wiki/teams/4605/wiki/experiment1.png)
3. Gently pipette up and down for several times to mix thoroughly (DO NOT VORTEX!). Centrifuge to collect the reaction solution to the bottom of the tube.
4. Incubate at 37℃ for 30 min and immediately chill the tube at 4℃ or on ice.

# Transformation

1. Thaw the competent cells on ice (e.g., DH5α Competent Cell, Vazyme #C502).
2. Pipette 10 μl of the recombination products to 100 μl of competent cells, flick the tube wall to mix thoroughly (DO NOT VORTEX!), and then place the tube on ice for 30 min.
3. Heat shock at 42℃ water bath for 45 sec and then immediately place on ice for 2 - 3 min.
4. Add 900 μl of SOC or LB liquid medium (without antibiotics). Then, shake at 37℃ for 1 h at 200 - 250 rpm.
5. Preheat the corresponding resistant LB solid medium plates in a 37℃ incubator.
6. Centrifuge the culture at 5,000 rpm (2,400 × g) for 5 min, discard 900 μl of supernatant. Then, use the remaining medium to suspend the bacteria and use a sterile bent glass rod to gently spread on the plate which contains the appropriate selection antibiotic.
7. Incubate at 37℃ for 12 - 16 h.

# Colony PCR

Vazyme | 2 × Rapid Taq Master Mix（P222）

### Materials

2 × Rapid Taq Master Mix

### Protocol

1. Prepare the reaction system

   ---

   ddH2O

   to 50.0 µl

   2 × Rapid Taq Master Mix

   25.0 µl

   Primer 1 (10 µM)

   2.0 µl

   Primer 2 (10 µM)

   2.0 µl

   Template DNA\*

   x µl

   ---

   Optimal reaction concentration for plasmid DNA is 0.1 - 10 ng.
2. Run PCR reaction

   a. The initial denaturation conditions are suitable for most amplification reactions and can be adjusted according to the complexity of the template structure. If the template structure is complex, the initial denaturation time can be extended to 5 - 10 min to improve the initial denaturation effect.
   b. The annealing temperature needs to be adjusted according to the Tm value of the primer, generally set to be 3 ~ 5℃ lower than the Tm value of the primer; For complex templates, it is necessary to adjust the annealing temperature and extend the extension time to achieve efficient amplification.
   c. For higher yields, the extension time can be set to 2 - 5 sec for PCR reactions with products less than 1 kb; for PCR reactions with products more than 1 kb, the extension time can be extended to 20 - 30 sec/kb.

# Agarose Gel Electrophoresis

### Materials

GelRed nucleic acid dyes
DL2000 DNA Marker
DL5000 DNA Marker
PCR products
Double digestion products

### Protocol

1. Add 1.2g of agarose and 80ml of 1×TAE buffer to a Erlenmeyer flask, and swirl to suspend the agarose powder in the buffer.
2. Place the gel solution into the microwave. Using a low to medium setting, set the timer for a minimum of 5 minutes, stopping the microwave oven every 30 seconds and swirling the flask gently to suspend the undissloved agarose.
3. Boil and swirl the solution until all of the small translucent agarose particles are dissolved.
4. Set aside the flask to cool to 60℃ before adding 2μl GelRed nucleic acid dyes.
5. Place the comb into the 15×7 cm slot of the tray and pour the molten agarose into the gel tray.
6. Allow 30-40 minutes for the gel to solidify at room temperature.
7. Carefully remove the comb from the solidified gel and remove the tape from the edges of the gel tray.Place the tray onto the leveled Sub-Cell so that the sample wells are near the cathode.
8. Submerge the gel beneath 2 to 6 mm of 1× TAE buffer.
9. Load the samples and the marker.
10. Close the lid of gel tank and apply a voltage of 75V.
11. When the DNA samples or dyes have migrated a suffcient distance through the gel, turn off the electric current and remove the leads and lid from the gel tank.
12. Place the gel on the UV transilluminator for nucleic acid visualization and analysis.

# Extract the plasmid

Vazyme | FastPure Plasmid Mini Kit（DC201）

### Materials

RNase A
Buffer P1
Buffer P2
Buffer P3
Buffer PW1
Buffer PW2
Elution Buffer
FastPure DNA Mini Columns
Collection Tubes 2 ml

### Protocol

1. Transfer 1 - 5 ml of overnight (12 - 16 h) culture to a centrifuge tube (not provided), and centrifuge at 10,000 rpm (11,500 × g) for 1 min. Discard the culture medium and place the tube inverted on a blotting paper to drain the liquid.
2. Add 250 μl of Buffer P1 (make sure that RNase A has been added) to the centrifuge tube containing the precipitated bacterial cells, and mix thoroughly by pipetting or vortexing.
3. Add 250 μl of Buffer P2 to the mixture from Step 2. Mix by gently inverting the tube 8 – 10 times to completely lyse the cells.
4. Add 350 μl of Buffer P3 to the mixture from Step 3. Immediately invert the tube gently 8 – 10 times to fully neutralize Buffer P2. At this time, white flocculent precipitates should form. Centrifuge at 12,000 rpm (13,400 × g) for 10 min.
5. Place FastPure DNA Mini Columns into a Collection Tube 2 ml. Carefully transfer the supernatant from Step 4 to the FastPure DNA Mini Columns with a pipette, taking care not to disturb the precipitates. Centrifuge at 12,000 rpm (13,400 × g) for 30 - 60 sec. Discard the filtrate and place the FastPure DNA Mini Columns back into the Collection Tube.
6. (Optional) Add 500 μl of Buffer PW1 to the FastPure DNA Mini Columns. Centrifuge at 12,000 rpm (13,400 × g) for 30 - 60 sec. Discard the filtrate and place the FastPure DNA Mini Columns back into the Collection Tube.
7. Add 600 μl of Buffer PW2 (make sure that absolute ethanol has been added) to the FastPure DNA Mini Columns. Centrifuge at 12,000 rpm (13,400 × g) for 30 - 60 sec. Discard the filtrate and place the FastPure DNA Mini Columns back into the Collection Tube.
8. Repeat Step 7.
9. Place the FastPure DNA Mini Columns back into the Collection Tube. Centrifuge the empty column at 12,000 rpm (13,400 × g) for 1 min to completely remove the residual wash buffer.
10. Place the FastPure DNA Mini Columns in a new sterile 1.5 ml centrifuge tube. Add 30 - 00 μl of Elution Buffer to the center of the spin column membrane. Leave the system at room temperature for 2 min, and centrifuge at 12,000 rpm (13,400 × g) for 1 min to elute the DNA.
11. Discard the FastPure DNA Mini Columns and store the extracted DNA at -20°C to prevent degradation.

# Preparation of Competent Komagataeibacter xylinus

### Materials

Komagataeibacter xylinus ATCC 700178
sterile pre-chilled water
sterile pre-chilled 10% glycerol

### Protocol

1. Inoculate the bacteria into 5ml of culture medium and cultivate on a shaker at 30°C for 2 days.
   The following steps must be carried out entirely on ice and within a clean bench.
2. Inoculate the cultured bacteria into 100ml of culture medium at a ratio of 1:100. Cultivate at 30°C with agitation at 180rpm until the optical density (OD) reaches 0.4-0.6.
3. Place the culture on ice for 30 minutes, then transfer it into 50ml EP tubes within the clean bench.
4. Centrifuge at 6000rpm at 4°C for 10 minutes and discard the supernatant.
5. Add 35ml of sterile pre-chilled water to resuspend the bacterial pellet.
6. Centrifuge again at 6000rpm at 4°C for 10 minutes and discard the supernatant.
7. Resuspend the bacterial pellet in 20ml of sterile pre-chilled water.
8. Repeat the previous step.
9. Suspend the bacterial pellet in 2.5ml of sterile pre-chilled 10% glycerol, and aliquot 150μL into 1.5ml EP tubes. Store at -80°C for later use.
   Calcium ions can be added for optimization during the preparation of competent cells.

# Electroporation

### Materials

ddH2O
75% ethanol
absolute ethanol
competent cells
recombinant plasmid

### Protocol

1. Clean the 0.2 mm electroporation cuvette with ddH2O, followed by 75% ethanol, and then absolute ethanol.
2. Thaw a certain amount of competent cells (approximately 40μL) on ice, add 6μL of recombinant plasmid (specific volume to be determined). After a 30-minute ice bath, transfer the mixture into the electroporation cuvette, taking care to avoid bubble formation.
3. Perform electroporation at 2.5kV for 5ms. In the clean bench, add 1ml of culture medium, then transfer the mixture into a 1.5ml EP tube and incubate at 30°C with shaking at 180rpm for recovery.
4. After 7 hours, centrifuge at 8000rpm for 1 minute, discard 900μL of the supernatant, resuspend the pellet, and streak onto a selective solid culture medium. Incubate at 37°C.

# Preparation of HS culture medium

### Materials

Glucose
Peptone
Yeast extract
Na2HPO4
Citric acid
Distilled water

### Protocol

1. Rinse the graduated cylinder and the large beaker three times with distilled water.
2. Prepare HS culture medium according to the following formula.

   ---

   Glucose

   20 g/L

   Peptone

   5 g/L

   Yeast extract

   5 g/L

   Na2HPO4 (Sodium hydrogen phosphate)

   2.7 g/L

   Citric acid

   1.15 g/L

   ---
3. Add water to reach 800-900ml.
4. Place it on a magnetic stirrer, stir, then transfer it to a graduated cylinder. Add water to reach 1L and pour it back into the beaker.
5. Briefly sonicate the solution to make it clear.
6. Distribute into two conical flasks, each with 100ml, and store the remaining 800ml in a glass bottle.
7. Autoclave at 115°C (including glucose, so it cannot exceed 115°C, as it may caramelize) for 20 minutes.

# Measurement of the K. xylinus growth curve

### Materials

bacterial culture medium
cellulase

### Protocol

1. Take 3 sterilized 250 ml Erlenmeyer flasks, labeled 1, 2, and 3, and add 50 ml of HS culture medium respectively.
2. Set the initial OD value of the bacterial solution to 0.02. The volume of bacterial culture medium to be added is calculated.
3. Add cellulase in the bottle at 1:1000, respectively.
4. Seal the Erlenmeyer flask, put it into a shaker for culture, and take out 3-4ml of bacterial culture medium every 3 hours to measure the OD value.

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{% block lead %}The entrepreneurship prize recognizes exceptional effort to build a business case and commercialize an iGEM project.{% endblock %}
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# 1. Successfully optimized the cultivation conditions for K.xylinus

![](https://static.igem.wiki/teams/4605/wiki/result/1.png)

Figure.1 Growth curve of K. xylinus

K.xylinus, unlike engineered strains such as Escherichia coli or Saccharomyces cerevisiae, are not extensively studied in terms of their growth conditions, culture media, and methods for competent bacteria preparation. Therefore, our initial focus was on determining the growth curve of K.xylinus.

We have observed that K.xylinus enter a plateau phase approximately 48 hours after cultivation. Based on the growth curve of K.xylinus, we can roughly determine that they are in the logarithmic growth phase between 15 to 25 hours.This information is valuable in determining the optimal timing for preparing competent K.xylinus, among other applications.

Initially, during cultivation, we noticed that both the growth rate and membrane production efficiency were significantly low. After conducting extensive literature research, we made attempts to adjust the glucose and ethanol concentrations in the HS culture medium. By measuring the dry weight of the BC membrane produced by K.xylinus, we analyzed the impact of these two culture parameters and their interaction. Furthermore, we collaborated with the dry lab to establish a model and delve deeper into determining the optimal culture conditions. Our model revealed that the highest membrane yield was achieved when the glucose concentration ranged between 30g/L and 35g/L, while the ethanol concentration remained within 0% to 0.5%. These findings align with the report by S.A. Hutchens et al.

![](https://static.igem.wiki/teams/4605/wiki/result/2.png)

Figure.2 The influence of different glucose concentrations and ethanol concentrations on the dry weight of BC membrane

![](https://static.igem.wiki/teams/4605/wiki/result/3.png)

Figure.3 Impact curve of BC membrane over time from the dry lab

We provide the following explanation:

The presence of ethanol or glycerol as alternative electron sources has been reported to repress glucose oxidation in K.xylinus. This repression allows for an increased yield of cellulose production, as glucose synthesis competes with glucose oxidation by the membrane-bound glucose dehydrogenase enzyme. Additionally, K.xylinus possess membrane-bound alcohol and aldehyde dehydrogenases, which can oxidize alcohol to acetic acid to generate electrons.

# 2. Successfully constructed a indigoidine biosynthetic pathway in C. glutamicum

![](https://static.igem.wiki/teams/4605/wiki/result/4.png)

Figure.4 Plasmid fragment of bpsA and the expression in C. glutamicum

SDS-PAGE showing resolve of Marker,

1: cell lysate of wild type C. glutamicum,

2: cell lysate of IPTG-induced C. glutamicum expressing bpsA,

3: supernatant of 1,

4: supernatant of 2.

Stained using Coomassie Brilliant Blue. bpsA predicted to be 140 kDa

Based on the NFLS\_Nanjing 2022 project, we utilized their composite part BBa\_K4376003 as a reference and introduced our composite part BBa\_K4605010 into C. glutamicum. The successful expression of indigoidine synthetase in C. glutamicum was confirmed through SDS-PAGE analysis. After induction with 1 mM IPTG for 12 hours, we observed a blue-colored bacterial pellet upon centrifugation, as depicted in Figure X. In contrast, when we introduced the empty vector pEKEX2, the fermentation broth appeared yellow, indicating the establishment of a metabolic pathway for indigoidine synthesis in C. glutamicum. Notably, the supernatant obtained after centrifugation in Figure X exhibited a light blue color, suggesting that C. glutamicum secretes a portion of the dye into the culture medium following indigoidine synthesis. Unfortunately, due to limitations, we were unable to perform transmission electron microscopy on the genetically modified C. glutamicum to visualize how the pigment is secreted out. However, this conclusion is supported by the electron microscopy images reported by Mohammad Ghiffary et al.

To further confirm the synthesis of indigoidine, we conducted scans and obtained the maximum absorption peak of the product, which coincided with the reported maximum absorption wavelength of indigoidine at 590nm. This indicates that our genetic modification and metabolic pathway construction in C. glutamicum were successful. We explored various organic solvents for extracting indigoidine and ultimately found that DMSO yielded the best results. By suspending the bacterial cells in DMSO, followed by sonication for cell disruption, we obtained the supernatant, resulting in the production of indigoidine microbial dye, as shown in Figure 5.

![](https://static.igem.wiki/teams/4605/wiki/result/5.png)

Figure 5. Blue pigment indigoidine secreted by IPTG-induced/pTac-ind C. glutamicum. The strain was induced with 1 mM IPTG at 30 °C for 12 h.

(A) A small amount of indigoidine was secreted into the culture medium, with the majority still remaining inside C. glutamicum.

(B) IPTG-induced/pTac-ind C. glutamicum with pEKEX2 (right) and IPTG-induced /pTac-ind C. glutamicum with pEKEX2-bpsA (left)

(C) bsorption spectrum of our product

(D) DMSO extraction of indigoidine

(E) Extraction of indigoidine produced by C. glutamicum using different organic solvents.

# 3. Successfully obtained a colored BC membrane that binds to liposoluble dyes through co-cultivation

![](https://static.igem.wiki/teams/4605/wiki/result/red.png)![](https://static.igem.wiki/teams/4605/wiki/result/yellow.png)

In a 20mL cultivation medium for BC membrane, different concentrations of carmine/lemon yellow dye were added. The mixture was incubated for 6 days, and then the BC membrane was removed and washed several times with water until no more color was observed. A 0.5g portion of the membrane was taken and added to 3mL of water. Then, a 30μL concentration of 3000U/mL cellulase solution was added for membrane degradation, resulting in a suspension. The suspension obtained from the colorless BC after lysis was used as a blank control. The absorbance value at 428nm (the maximum absorption wavelength of lemon yellow) was measured.

![](https://static.igem.wiki/teams/4605/wiki/result/6.png)

Figure.6 Water-soluble dye staining results: carmine & lemon yellow

In order to validate the binding performance of BC membrane with dyes, we first added different concentrations of carmine and lemon yellow into the cultivation medium of K.xylinus to obtain the relationship between water-soluble dye concentration and OD value. As shown in Figure 4, this method allowed us to obtain uniformly stained BC membrane. However, the results were not as expected, as the color of the BC membrane did not become darker with increasing dye concentration. On the contrary, there was no clear linear relationship observed, and the relationship between the two followed a bell-shaped curve with a peak. This indicates that higher concentrations of water-soluble dyes can exhibit side effects, such as the inhibitory effect on the growth and membrane production of K.xylinus, as observed when adding 10mg/ml of carmine to the cultivation medium. This indicates that even with water-soluble dyes, direct staining can still pose issues with high dye concentrations affecting the growth of K.xylinus.

The effect of indigoidine (DMSO extracting) dyeing is not very good either, uneven and the color is lighter. Therefore, after reviewing the literature and brainstorming, we decided to obtain the target product by co-culture method, that is, to culture indigoidine-expressing and BCM-expressing bacteria together, in order to make the fat-soluble dye dyeing effect better.

![](https://static.igem.wiki/teams/4605/wiki/result/7.png)

Figure 7.carmine (left), lemon yellow (middle) and indigoidine (right) staining result

![](https://static.igem.wiki/teams/4605/wiki/result/8.png)

Figure 8. shaking cultivation of the genetically modified C. glutamicum and BC membrane-producing K.xylinus

Electron microscope of the indigoidine-dyed BC particles:

magnification=5000x (left) magnification=10000x (right)

Co-culture results of the first step: We conducted shaking cultivation of the genetically modified C. glutamicum and BC membrane-producing K.xylinus as described above. We obtained the production of relatively uniform blue BC particles. To further illustrate the interaction between the BC membrane and C. glutamicum during co-cultivation, scanning electron microscopy (SEM) was employed. The SEM images provide visual evidence of the successful co-cultivation process, demonstrating the binding and integration of BC membranes with C. glutamicum.

These findings support the successful co-cultivation of the modified C. glutamicum and K.xylinus for the production of BC membranes. Microfibers produced by K.xylinus intertwine with each other to form a mesh-like structure, in which the indigoidine-secreting C. glutamicum are encapsulated. Due to the blue color of the genetically modified C. glutamicum, the BC particles that encapsulate them also appear blue. However, this deviates from our initial goal of producing a solid BC membrane that displays a distinct blue color and is applicable in the fashion industry. To make it suitable for production purposes, further processing is required. For instance, we may need to compress the BC particles into a membrane using a high-pressure filtration method.

![](https://static.igem.wiki/teams/4605/wiki/result/9.png)

Figure 9. Inoculating genetically modified C. glutamicum on the BC membranes as a scaffold

Co-culture results of the second step: Based on extensive literature research, we have discovered that BC membranes are highly biocompatible scaffold materials used for culturing cells. Initially, we successfully obtained a complete BC membrane by cultivating K.xylinus. Subsequently, we placed the BC membrane on a culture medium containing C. glutamicum and conducted line cultivation, which resulted in the production of indigoidine. As a result, we successfully obtained blue BC membranes with custom-designed patterns. This method, in comparison to the co-culture results of the first step, offers a complete membrane structure and allows for independent creativity based on the producer's ideas. Most importantly, it accomplishes our initial goal of obtaining indigoidine-dyed BC membranes without the need for optimizing the culture medium.

# 4. Successfully constructed plasmids for expressing chromoproteins or synthesizing indigoidine in BC-producing strains—— K.xylinus

![](https://static.igem.wiki/teams/4605/wiki/result/10.png)

Figure 10. Gel electrophoresis of palsmids for expressing chromoproteins EGFP/YFP in K.xylinus

What we are working right now:

The fluorescence per cell would be measured using flow cytometry in K.xylinus strains that expressed EGFP/YFP under the control of three different heterologous promoters J23100, J23104, J23119. We can also use the plasmids we built to see how cellulose wraps up K. xylinus. Most importantly, chromoproteins can be used to characterize promoter strength so that we could add more information to K. xylinus genetic toolkit. We are currently working on expressing chromoproteins in K.xylinus to obtain cellulose membranes that emit fluorescence. This could potentially be used in the fashion industry in the future.

![](https://static.igem.wiki/teams/4605/wiki/result/11-1.png)![](https://static.igem.wiki/teams/4605/wiki/result/11-2.png)

Figure 11. Gel electrophoresis of palsmids for expressing indigoidine in K.xylinus

Meanwhile, we are also attempting to reconstruct the metabolic pathway for indigoidine synthesis in K.xylinus. We have referenced the existing knowledge on indigoidine synthesis in other bacteria, such as C. glutamicum. We would also take into consideration the expression results of chromoproteins in K.xylinus as a reference to fine-tune our promoter.

# Future work

Once we have finished the construction of the color fiber system, the metabolic pathway for other liposoluble dyes, such as violacein, Tyrian purple, melanin and indigo can also be introduced into K.xylinus. We have conducted research on the synthesis pathways of these dyes (Yang, D. et al. 2021)and found that the process requires relatively few enzymes, making the construction of a metabolic system feasible. This approach would provide a paradigm for effectively addressing pollution issue in fashion industry and bring synthetic biology and its concepts to the public. Moreover, it holds immense potential for future production and application.

# References

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[2] Dyes ACS Sustainable Chemistry & Engineering 2021 9 (19), 6613-6622 Fricke, P.M., Klemm, A., Bott, M. et al. On the way toward regulatable expression systems in acetic acid bacteria: target gene expression and use cases. Appl Microbiol Biotechnol 105, 3423–3456 (2021).

[3] Goosens VJ, Walker KT, Aragon SM, Singh A, Senthivel VR, Dekker L, Caro-Astorga J, Buat MLA, Song W, Lee KY, Ellis T. Komagataeibacter Tool Kit (KTK): A Modular Cloning System for Multigene Constructs and Programmed Protein Secretion from Cellulose Producing Bacteria. ACS Synth Biol. 2021 Dec 17;10(12):3422-3434.

[4]Florea M, Hagemann H, Santosa G, Abbott J, Micklem CN, Spencer-Milnes X, de Arroyo Garcia L, Paschou D, Lazenbatt C, Kong D, Chughtai H, Jensen K, Freemont PS, Kitney R, Reeve B, Ellis T. Engineering control of bacterial cellulose production using a genetic toolkit and a new cellulose-producing strain. Proc Natl Acad Sci U S A. 2016 Jun 14;113(24):E3431-40.

[5]Teh MY, Ooi KH, Danny Teo SX, Bin Mansoor ME, Shaun Lim WZ, Tan MH. An Expanded Synthetic Biology Toolkit for Gene Expression Control in Acetobacteraceae. ACS Synth Biol. 2019 Apr 19;8(4):708-723.

[6]Mohammad Rifqi Ghiffary, Cindy Pricilia Surya Prabowo, Komal Sharma, Yuchun Yan, Sang Yup Lee, and Hyun Uk Kim.High-Level Production of the Natural Blue Pigment Indigoidine from Metabolically Engineered Corynebacterium glutamicum for Sustainable Fabric DyesACS Sustainable Chemistry & Engineering 2021 9 (19), 6613-6622

[7]Gilbert, C., Tang, TC., Ott, W. et al. Living materials with programmable functionalities grown from engineered microbial co-cultures. Nat. Mater. 20, 691–700 (2021).

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