Glowing Plant End of Year update
Sorry that this update is a little delayed, we wanted to wait until the results of some genotyping/PCR experiments we need to run were complete but we are still having trouble with those as we detail below.
Unfortunately without those results we don't have much of significance to report on the Glowing Plant except that we have now 30 transformed plants. We do however have better news to report on the Fragrant Moss which is progressing excellently. We remain optimistic that sales of the Moss will allow us to keep working on the glowing plant.
Glowing Plant Transformations
We currently have 18 plants transformed with our full construct and another 12 plants transformed with a reduced construct with just Lux CDE, which we made to overcome the issues we were having with Lux C in the full construct. This shows that our transformation process is working well.
Here are some images of the plants, you'll notice you can see 'bite marks' in some of the leaves. This is where we took tissue from the plant for genotyping. Because the genotyping has been going on so long these plants are bigger than they would normally be at the stage we check for the presence of trans-genes.
None of these plants glow, which isn't a surprise given the broken constructs we observed and detailed in our previous updates. Our hope however is that out of all the plants there is a combination of two plants which have a full complement of the genes so that we can cross breed them to get an offspring with the full complement of genes which we hope will glow.
When making a transgenic plant, there are several steps required to confirm the presence of the transgene. First, you check for the desired trait, which for us is luminosity. Unfortunately, none of the plants transformed with the full lux construct glowed (as expected given our earlier results).
The next step for confirmation is genotyping. Normally, when you know the site of insertion, primers are designed to amplify the border of the transgene and the plant genome (Figure?). Using a computer we design a DNA sequence which is complementary to the DNA in our target gene in the template, as illustrated by this figure:
We then synthesize the single strand complementary DNA which is called a primer, this is a common service we outsource. In San Francisco a driver comes by with the primer the next day, it's kind of like uber for synthetic DNA!
Since bombardment is not a targeted process but the DNA is randomly inserted across the tobacco genome, we cannot easily identify where the insertion is for each independent line. This is possible with extra time and effort using genome sequencing or inverse PCRs but that isn't normally worth it for us.
The easiest way to confirm an insertion of a randomly inserted transgene is using genotyping PCR with primers specifically designed to amplify the transgenic DNA we inserted. In any type of PCR, it is very important to include negative controls to make sure the bands appearing are specific to our experiment and that it worked correctly. We normally use two negative controls- a reaction with no template (ie fresh water!) and a reaction with wild-type tobacco gDNA. Both reactions should appear blank when the gel is run after the PCR. For those who are unfamiliar with the process of PCR, here is a video on youtube explaining the process (we didn't make one as this is such a common process):
And here's what it looks like in action on the lab bench:
Unfortunately our genotyping experiments have failed multiple times over the past six weeks. This has been extremely frustrating for the whole team, as this is really bread and butter basic lab work. During the genotyping process, we have encountered a nasty contamination in one or more of our PCR reagents. This led to a continuous appearance of a band in the negative controls. After multiple attempts clearing this contamination, which eventually required replacing all the materials and equipment we use, we finally are contamination free and now ready for the actual genotyping PCR. As we need to confirm the presence of multiple genes in 30 independent lines, it will take us some more time to have the data ready. We will follow up shortly with the results in the next couple of weeks.
Just in case we find that Lux C has a developmental issue with tobacco, we have started a new side project to create Glowing Moss. As we discuss below, we are pretty confident in all our processes from transformation to scale up with the moss and because it has a very different development process than tobacco it's unlikely that the same issues replicate themselves.
The central challenge with Glowing Moss is the number of promoters we require. Because our moss can perform homologous recombination this means we need a distinct promoter for each gene (though we are also testing methods to link the genes to reduce the number of promoters). Transgenic moss is a relatively niche field, and academic groups tend to just use the same few promoters each time because they are known to work. We can't use many of those (eg 35s) in our work for reasons related to USDA regulations. Monocot promoters work in moss, so there are plenty of available promoters to choose from, but we have to find the good ones. This means the first step is testing a broad range of these using our existing Flux assays, then we'll chose the ones that work and start the transformations. We can only insert two genes at a time, so this will require at least three rounds of sequential transformation which will take at least 6 months, then it takes 6-9 months to grow enough moss to ship and distribute so don't expect fast results from this.
Glowing Moss wouldn't be exactly the same as a glowing plant, but in some ways it would be more interesting because you can do more creative things with it, for instance you could make glowing moss graffiti covering your wall like this:
Work on getting the Fragrant Moss ready for shipping has not been without it's hiccups, as usual with biology we had unanticipated problems nearly every step of the way, but the team persevered and we are finally confident that we've solved all the remaining technical challenges and are on track for shipping around March.
The first challenge was scaling up the production of the moss so that we can grow really large volumes of it in order to ship out to the world. Initially we planned to grow in liquid culture, and that kind of worked but we found the moss grew much better on a solid media once we dealt with the contamination issues. There wasn't one single solution to the problem of contamination, it was lots of process improvements as well as upgrades to some of our equipment. Nevertheless we cracked it and the moss is now doubling in volume every 2-3 weeks. Our plan is to get to a steady state with five hundred customers/plates worth of moss, which will give us a production capacity of around 1,000 customers a month. The following photos show you how we are doing so far. Not all of these plates are production quality moss, some are other experiments, but we have about 150 plates at the moment so we are nearly at production scale.
Note that this is not what the moss will look like when it's grown, we keep it in this mat phase of growth (known as filamentous) as it has the fastest cell division rate. Later (or sooner with the addition of the right hormones) the moss moves into leafy growth. Then it will look like this:
Once we figured out how to grow the moss at volume in our lab we had to figure out how people are going to grow it at home. Because of the contamination issues we mentioned earlier we can't use the media we use to grow the moss in the lab as it also allows mold to grow if you open it to the air (which we want to do!). We also want it to look good of course. Figuring out what to grow the moss on turned out to be much harder than we expected. The main challenge was finding the right balance between keeping the moss damp enough and it getting to wet, which promoted contamination.
In the end we tested over 20 different materials. We have found that it is very important to grow the moss on a substrate which can retain moisture, since our moss prefers a wet growth environment. Sandstone samples from a few suppliers (including some very cool 3D printed shapes) seemed to perform well for this, at least at the outset, but often got overrun by fungus or required watering too frequently to prevent the moss drying out. We also tested several materials that are growth biologically, but all of these retained too much water and decomposed or grew mold.
The best candidate in the end was a clear soil substitute, it looks just like the agar we grow our moss on in the lab but moss grows on it and mold doesn't. Lately, we have experimented with adding salts and micronutrients to the clear gel soil substitute in order to boost growth and maintain the moss in its healthiest possible state, while minimizing watering requirements. Those experiments are ongoing but the basic material works well.
Having selected the growth media we had to choose the design for how it's going to look as well as the 'out of the box' and shipping experience. We were invited to demo the moss at Biofabricate in New York, and were lucky enough to have some designers from Parson's School of Design collaborate with us for that. This is what they came up with:
These designs look really cool, and we liked the nod to the science in the designs, but we also explored other shapes and based on focus group feedback ended up going with a terrarium style. The air-hole increases humidity inside the bulb and also allow the fragrance to build up a little more which is a nice feature. Here are some of the designs we are exploring, let us know which you like the most in the comments section below:
The last technical challenge was ensuring that we can ship the moss in the mail and have it arrive healthy and happy in the customers hands. We hoped to ship in liquid form so that people can make their own designs at home, but when we came back from the holidays we discovered that it grows too slowly from this stage so that won't be a good user experience. Instead we are testing a large number of additives and modifications to the growth media to see if we can kickstart the growth. We are also testing shipping the adult moss, those samples are in the mail this weekend and hopefully they come back to us happy after spending the long weekend with USPS. The adult moss would have the nice feature that it will smell out of the box (if it's happy in the mail!).
We have three tasks left to complete before we can start shipping, first we have to design suitable packaging that fits the product and means the glass won't break in the mail. Second we have to wait for delivery of the glass terrariums. And third we have to wait for the moss to go through a few more doublings. Currently we anticipate a March shipping date.
As well as all the work in the lab James Anderson-Furgeson spent three weeks at the end of October with our collaborators at the Danish Technical University improving our knowledge of how to grow and transform moss. He performed two transformations with the experts at DTU to improve his techniques. The transformation process involves digesting the cell wall of moss cells to form protoplasts, which are very delicate. The tricks of the trade that James learned in Copenhagen involve keeping those delicate protoplasts intact and healthy in order to increase transformation efficiency, and knowing which media and wash solutions to make fresh for each transformation, vital details that aren't found in research papers. One of the quirks of working with biology are the small details which can make a big difference to experiment efficiency and which are often not described. He also learned about how best to follow the growth of regenerating protoplasts in order to learn early whether the transformed moss survived the transformation. Learning that the protoplasts died early on in the regeneration process indicates that the transformation should be re-attempted immediately, speeding the process of creating a new moss line. This relationship also helped us improve our communication with the lab at DTU so that we have a resource in case technical issues arise in the future.
Finally we are looking ahead to future fragrant moss lines. We've made a version of the Patchouli moss which has an even stronger fragrance than our current moss line, we only have a tiny amount of this however so will take time to scale production. We have synthesized three more terpene synthase genes to transform into moss, and are designing expression constructs to create further fragrant lines. We also applied for grant funding to support the development of future fragrant moss lines and scale up our systems.
As we start yet another year of work on the project, I just want to thank all of you for your continued patience and words of encouragement. Like you we didn't expect this to take this long, each setback and challenge is felt by the team but it's really your ongoing support which keeps us going, especially in the face of criticism we get from time to time. Hopefully 2017 is the year we crack this.
Antony & the glowing plant team