Our project aims to boost bacterial cellulose (BC) production in Komagataeibacter xylinus and utilize it as a water-retaining substrate to enhance seedling survival rates in desert environments, addressing the issue of desertification. Genetic manipulation of Komagataeibacter xylinus is relatively challenging. Through continuous failed attempts, we found solutions. These optimized cultivation and DNA transformation protocols can serve as a reference for future iGEM teams working with this bacterium, helping to reduce exploration time. This includes optimizing growth conditions, successfully transforming DNA, and methods for multiplex genetic manipulation. Additionally, our interactive videos for education and safety training have received excellent feedback, offering a unique reference for designing educational activities. We have achieved the following contributions.

Solving the issue of DNA transformation

At the same time, due to the thick cell wall of Komagataeibacter xylinus, successfully transforming exogenous DNA is relatively challenging. After multiple attempts, we found that the medium used for transforming Komagataeibacter xylinus must include cellulase to degrade the bacterial cellulose encasing the bacteria; otherwise, the transformation efficiency would be significantly reduced. This may be because the bacterial cellulose interferes with the effectiveness of electroporation. The optimal transformation procedure is as follows: use a 0.1 cm sterile electroporation cuvette, apply 1.8 kV for 2.5 seconds each time, and deliver two pulses.

Wild-type Komagataeibacter xylinus has an effective restriction modification system that recognizes and degrades exogenous DNA, thus causing the exogenous plasmid not to be stable in this bacterium. To solve this problem, we have learnt through communication with experts that it is possible to perform genetic manipulation directly on the genome of Komagataeibacter xylinus by recombination, which will lead to stable inheritance. However, gene editing of multiple genes suffers from too little selectability of resistance genes.

In our project, for the overexpression of the pgi gene from E. coli, the sacB locus, which encodes levansucrase, was selected as a homologous recombination site First, the kana expression cassette will replace the sacB gene locus as a selectable marker. Then, pgi will replace the kana gene, resulting in the loss of kana resistance, confirming successful gene insertion.

To knock out the gdh gene based on the pgi-OE strain, we introduced the kana gene into Komagataeibacter xylinus. Specifically: the kana gene will be inserted at the gdh locus in K. xylinus to achieve gene knockout. To facilitate screening for positive transformants, the kana gene will be expressed under the control of the tac promoter and rrnB-T terminator. Overall, Komagataeibacter xylinus will undergo a kanamycin resistant - non-kanamycin resistant- kanamycin resistant, resulting a pgi-OE gdh-KO strain.

Using online interactive science communication methods enhances engagement and promotes active learning while catering to diverse audiences. This approach allows for real-time feedback, fosters curiosity, and simplifies complex concepts. Additionally, it increases audience participation and helps improve the retention of scientific knowledge, ensuring that content is effectively remembered by viewers. Compared to traditional lecture-style videos (right), interactive videos clearly have a higher viewership (left).

Therefore, we hope to share this innovative approach with future iGEM teams, enabling them to build upon our foundation and make further innovations and iterations.

Here is the link to the full video we created: https://b23.tv/ei8YnWr

Step 1. Prepare the Video Content
You will need to create separate video clips for each possible option, ensuring that each choice has a clear branching storyline. Make sure the clips are self-contained and logically connected.

Step 2. Apply for Interactive Video Permissions
Currently, Bilibili’s interactive video feature is not automatically available to all users. You will need to apply for permission through Bilibili’s "Creator Center":

• Log in to your Bilibili account and enter the "Creator Center."
• In the "Feature Settings" section, find "Interactive Video" and submit an application. Wait for approval.

Step 3. Upload Video Materials
Once you have the necessary permissions, go to the interactive video editing interface:

• Upload all the prepared video clips. Make sure each clip follows the correct order or logic.

Step 4. Create Interactive Nodes
Using Bilibili's interactive video editor, you can add interactive nodes where viewers can make choices.

• Add Nodes: Insert interactive nodes at points where you want viewers to make a decision.
• Set Options: For each node, add multiple options and ensure each option links to the corresponding video clip.
• Branching Logic: Set up the branching logic through the editor so that viewers are directed to different storylines based on their choices.

Step 5. Test and Preview
After setting up the initial structure, use Bilibili's preview function to test the video. Ensure that every branch and interactive node works correctly and that viewers are directed to the appropriate video segments based on their selections.

Step 6. Publish the Interactive Video
Once all the branches and interactive nodes are set up and tested, you can publish the video. Be sure to mention in the title or description that it is an "interactive video" to encourage viewers to engage with it.

Step 7. Analyze Data and Optimize
Bilibili provides data analytics tools to help you track viewer choices. Based on these insights, you can further refine and optimize your branching storyline design to increase engagement.

By following these steps, you can create an interactive video on Bilibili, allowing viewers to choose different options and experience varied storylines.