Glowing Plant June update & contest to win first Fragrant Moss
It's been a while since we did an update on the Glowing Plant science, so here one is. We haven't stopped working on the plant, we've been mostly focused on debugging the issues around transformation. We've now figured out the problem and have a clear plan for how to fix things, which we detail below.
We would like to thank all of you who are supporting our Wefunder campaign, which has now reached the minimum investment level. We have also completed science work on the fragrant moss so are hopeful this can help fund further glowing plant research - more on that below (or at this link), including information on how to get early access to the moss, as well.
Issues with construct bombardment
As we mentioned in our previous updates, we had lack of success from any of the regenerated plants from the first batch and have been testing our hypotheses of what's going wrong to understand why.
Our first hypothesis was premature removal of selection pressure leading to false positives and seedling. So our first step was keeping the transformed tissue longer on selection media which led to delayed regeneration and a dramatic reduction in the number of regenerants. This result supports our hypothesis that majority of our previous regenerants were false positives. Now that some of our regenerants finally rooted, we assayed the luminosity and were disappointed to find out that they still didn't glow.
This gel photo shows genotyping results for the Hygromycin cassette and luxA, B and C in three independent regenerants. The fourth lane for each gene is a positive control- the plasmid used for bombardment as a template. As you can see, Hygromycin cassette and luxA and luxB were present while luxC were not. We know that the PCR worked for luxC since the positive controls worked. This result strongly suggests that the insertion cassette broke or rearrangement occurred between luxB and C during the process.
There are multiple steps in the process that can lead to a broken cassette. 1. Since we are dealing with a large DNA molecule (over 15kbp), the gel purification step after the linearization can lead to a damage in the DNA. We make sure the DNA is intact after the purification by checking on a gel. 2. In case the linear DNA was fragile for some reason, coating on gold particles and the actual bombardment can lead to damage in DNA as this applies strong mechanical force. 3. The DNA gets broken or rearranged during the process of genome integration. Considering the fact that three regenerants tested all come from the same batch of bombardment and that they all show lack in luxC suggest that the lux DNA cassette broke during earlier steps in the process. Since we confirm the DNA after purification, it is highly likely the damage occurred during the bombardment.
In order to address this, we checked the effect of coating and bombardment by running an onion bombardment with the lux cassette in a circular versus a linear form. The linear DNA still works and produces luminosity in onion cells but there is a noticeable difference between the circular and linear DNA.
The amount of DNA used for coating was calculated to keep the starting amount of cassette equivalent between the two constructs, so the difference in luminosity likely refers to the lower efficiency of the linear cassette and part of this can be due to DNA getting destroyed (or broken) during the process.
There are several ways we can overcome this challenge.
1. Since the onion bombardment shows that the linear cassette still works albeit at lower efficiency, it could be a number's game - if we do more transformations eventually one will work. Hence we will be pushing our bombardments routinely until we get a plant that glows. Also, we still have a several more regenerants from earlier batches of bombardments waiting to be tested so we will see if any one of those glow.
2. To increase the efficiency of bombardment we will try two different methods in parallel. First, we will be dividing up the full lux cassette into two pieces (i.e. luxA, B, G and luxC, D, E) then bombard each separately. After retrieving plants either with luxA, B, G or luxC, D, E, we will cross the two lines to generate a plant with the full lux cassette. This will involve a decent amount of crosses and screening but we think smaller DNA cassettes have better chance to survive bombardment and safely integrate into the genome, and anyway we have to do crosses to remove the herbicide genes. The second approach involves eliminating co-bombardment by using a cassette which includes a selectable maker gene, but one that we don't need to outcross. This approach still has to deal with the stability of large linear DNA but we believe the efficiency will increase by doing a single DNA insertion and would be beneficial when playing number's game as we don't have inefficiency from the co-bombardment. We have identified a new selectable marker for this. Up to now, we have been using hygromycin as our selectable marker, this is a herbicide and therefore not suitable for environmental release. Our new marker is based on different conformation of amino acids. For each amino acid, there is a L- and D-type conformation. Normally, depending on species, one of the conformations are used while the other is toxic. We have found a gene that converts the toxic type to the beneficial type in plants, which in turn when transformed into a plant, can rescue the plant from growing on a media supplemented with the toxic form of amino acid. We have confirmed that this selectable marker can be used for our bombardments by confirming the toxicity of the amino acid by growing tobacco and Arabidopsis on them. We also confirmed rescue of transformed Arabidopsis on the toxic media. This selectable marker cassette has been added to our lux construct and now ready to be bombarded into plants.
3. A third option is to create a synthetic operon, and link all the genes to a single promoter. This approach has been shown to work to make glowing human cells (seriously!). Our early experiments with this approach failed but we are going to try some alternative linker sequences, if this works it should reduce the construct size by 50%.
Next, we would like to talk about our genetically engineered moss. We have recently announced our first Fragrant moss has passed all the technical challenges and are waiting to be bulked up. We are now in the process of bulking up production of this moss so that we have enough to distribute to customers. We are also exploring different planter designs. Here's a video showing our previous strain of fragrant moss being demoed to some random people in San Francisco:
Our hope and expectation is that this moss will be a commercial success (people certainly seem to like it!) and that we can use the proceeds from selling the moss to keep funding our Glowing Plant work, as we've discussed in previous updates.
Several of you have asked where you can get the moss from - we still need to bulk up production of the so we have enough to sell. In the meantime though we are running a contest for the first four people in the world to get access to this moss. You can enter that (free) contest by clicking on this link.
In addition to Fragrant Moss we are exploring making a Glowing Moss. The process is not simple for several reasons. First, we cannot use the current construct since the dicot promoters do not work in moss. Also, the moss uses homologous recombination to integrate the transformed DNA into its genome which is different to higher plants as plants rely more on random insertions. Homologous recombination can be a huge advantage when dealing with small number of parts as we can directly target a loci in the genome to eliminate positional effects and it also allows us to assemble constructs in vivo. The efficiency is also known to be higher than random insertions. But with a six gene pathway for autoluminescence, we will need more promoters/terminators to prevent recombination occurring within our parts which would delete the inserted genes. There are some methods we can look into to overcome this challenges and we see a nice potential in the Glowing moss so stayed tuned for future updates on this.
Update on financial situation
Now that our WeFunder campaign has passed it's minimum we are passed the financial crisis point we talked about in March. The current level of WeFunder funding should be enough to get the Fragrant Moss to market, but probably isn't enough to complete the Glowing Plant given the ongoing issues we are debugging. Our expectation is that funds from selling Fragrant Moss will get us to the point we are shipping the Glowing Plant - I'm really sorry this is taking so much longer than we and you wanted.
Till next time,
Antony & the Glowing Plant team