Anthony Tan
Anthony Tan
Plant Engineering Lab Notebook
A snapshot of synthetic biology experiments, lab techniques, and plant engineering protocols I performed while developing teaching materials for my classes. Open-source for educational purposes.
July 2024 - present
Plant Genetic Engineering Workshops
I teach students agrobacterium-mediated transformation. It has been nearly two years since I developed my own curriculum which I have taught dozens of times around the Bay Area, most notably at Berkeley City College.
These lab entries cover experiments for the new content I am developing.
To start off, Agrobacterium is a naturally-occuring pathogenic bacterium that has the ability to transfer genes into plants. Agrobacterium genes hijack the plant's machinery to produce food and shelter for the bacteria.
The key components for gene transfer are found on its T-DNA plasmid. If we swap out the parasitic genes with genes of interest, we can effectively engineer plants!
September 4, 2025
Inspiration for new content
I taught a workshop at BioPunk and told attendees about Light Bio's glowing petunia plant, which they immediately became excited about.
One participant asked, "Can we create a glowing plant?" Up until then, I had only been using RUBY red pigment construct as a teaching reporter.
Their curiosity and excitement sparked my interest in creating glowing DIY plants that they could enjoy.
[Photo I took of Firefly Petunia at BioCurious Community Lab in Santa Clara.]
OCTOBER 2025
Glow from Fluorescent Proteins
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Many fluorescent proteins such as GFP are largely invisible under ambient lighting and brilliantly bright under blue light
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A reporter that allows us to easily screen plantlets while viewing for extended periods of time is critical for future tissue culture dissections
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Super-Folder GFP (sfGFP) has a high extinction coefficient and can be excited by blue light, making it safe to look at. It carries no risk of UV-induced vision impairment unlike other GFPs
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As shown here, E. coli colonies expressing sfGFP are colorless under ambient light and bright green under blue light
October 2025
Plasmid Design
Many T-DNA plasmid backbone templates exist. Popular ones are from the pCAMBIA series. I chose pCAMBIA 1300 for a number of reasons, primarily:
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It confers plant resistance to hygromycin (hygR), which is better than kanamycin for robust transgenic cell selection in petunia and tobacco.
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It doesn't have unecessary reporter genes like GUS reporter.
A colleague of mine had cloned in a Arabidopsis UBQ10 promoter, a 1kb insert, and a UBQ3 terminator into pCAMBIA 1300. This promoter is favorable for future stable transformation projects compared to the standard Cauliflower Mosaic Virus 35S promoter because Arabidopsis promoters are less likely to be transcriptionally silenced by the plant over time.
He gifted me his construct and I decided to build off it. The task at hand is to remove my friend's 1kb insert and clone our desired sfGFP into the plasmid.
NOVEMBER 2025
Molecular Cloning
There are many types of cloning strategies. Let's focus on two popular ones:
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Gibson Assembly - utilizes homologous regions for annealing
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Pros: simple to design and effective at assembling a few fragments
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Cons: addition of arms of homology requires long primers ($)
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Golden Gate - utilizes Type II restriction enzymes for unique sticky ends
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Pros: can handle complex assemblies and uses shorter primers
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Cons: requires domestication/removal of unwanted restriction sites
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I chose gibson assembly for simplicity and designed the appropriate regions of homology between the plasmid backbone and the sfGFP insert. I used inverse deletion PCR to generate a linearized backbone without my friend's 1kb insert. I also did a DpnI digest to remove the unwanted template plasmid which could interfere plasmid selection given that both plasmids contain the same antibiotic resistance gene and can't be distinguished from selection alone.
Then I PCR'ed the sfGFP insert from another construct using primers that contained an overlap to the UBQ10 promoter and UBQ3 terminator. After PCR cleanup, I performed a gibson assembly reaction on the two DNA fragments.
December 2025
Plasmid Propagation & Colony PCR
Now that the sfGFP plasmid has been assembled, it needs to be propagated. We can use the biological machinery of fast-growing E. coli to produce thousands of copies of plasmids overnight
I heat shocked E. coli with the plasmid and plated the cells on petri dishes with antibiotic based on the bacterial antibiotic resistance gene on the plasmid
Next morning, I harvested some cells from multiple colonies and performed a diagnotic colony PCR & gel to check which cells contain sfGFP (lower bands)
Colonies with promising results were grown in liquid culture and miniprepped to harvest plasmids. Plasmids were sequenced to verify identity of plasmid
January 7, 2026
Transient Expression - Success!
I electroporated the T-DNA plasmid into GV3101 Agrobacterium tumefaciens, which contains a helper plasmid with virulence genes that faciliate T-DNA gene transfer into plant cells. These two plasmids use different antibiotic selection
I grew up the custom GV3101 sfGFP strain I made and resuspended the cells in infiltration buffer with acetosyringone to induce virulence. Then I injected the bacterial solution into a petunia petal.
Within days, injected cells accumulated significant amounts of sfGFP and glowed under blue light. Next up is creating a bioluminescent plant! Stay tuned!
Creating a Transgenic Plant
We validated some plant gene expression constructs. What now? How do we bring a GMO plant into the world?
November 2025 to Present
Stable Transformation
Pictured here is a an entire tobacco plantlet expressing red beetroot pigment.
[Green cells escaped herbicide selection due to limited exposure to media.]
The panel of images below demonstrates my process for generating stable transformants that can pass down heritable GMO traits when propagated
First, seeds are sterilized and grown in-vitro.
Next, sterile leaves are co-cultivated with agrobacterium.
Third, agrobacterium is killed with antibiotics and leaves are grown on callus induction media to stimulate stem cell proliferation.
Lastly, transformed tissue is dissected and plated on shoot induction media with herbicide to maintain cells with GMO and herbicide resistance genes