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Create GLOWING PLANTS using synthetic biology and Genome Compiler's software - the first step in creating sustainable natural lighting
Create GLOWING PLANTS using synthetic biology and Genome Compiler's software - the first step in creating sustainable natural lighting
Create GLOWING PLANTS using synthetic biology and Genome Compiler's software - the first step in creating sustainable natural lighting
8,433 backers pledged $484,013 to help bring this project to life.

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The End of the Road

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Orbella Fragrant Moss is Shipping!

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Hello,

I'm excited to announce we have finally started shipping Orbella Fragrant Moss. In this update we've got some more information on the product, information on how to use your credit from backing this Kickstarter to get one and an invitation to a party we are having on August 3rd to celebrate this milestone.

Introducing Orbella Fragrant Moss
Introducing Orbella Fragrant Moss

Thank you for your patience over all these years. For the team and I this is a major milestone on a journey that has been long and full of setbacks. We would never have got here without your backing this Kickstarter all those years ago and showing the world that biotechnology doesn't have to be done by big companies or major research institutions. 

I know some of you will be disappointed that this isn't glowing yet, and I'm sorry for that. This is the closest thing to that we've been able to make so far. Orbella represents many of the same values: it's a symbol of a future where we engineer living things for new functions. Biology is the ultimate sustainable technology, nature uses sunlight, CO2 and water to create useful products creating only biodegradable waste. I hope that Orbella can positively change the public’s perception of GMOs and inspire a new generation of bioengineers to create a greener future for us all.

As we've discussed in previous updates the plan is to use the profits from selling this moss to keep working on the glowing plant project. One day we will finally crack it! In the meantime you can apply the amount you backed this project as a credit to get the moss, full details below.

About Orbella Fragrant Moss

As you may remember from previous updates Orbella will ship in a glass terrarium, and once you set it up it will look like this:

Orbella Moss
Orbella Moss

The benefit of Orbella over traditional home fragrance products is that we don't have to dissolve the fragrance in a container. Typically containers are solvents or other petroleum based materials (eg wax) which evaporate into the air along with the fragrance. Orbella is cleaner – the fragrance is made right there in situ in your home from sunlight, CO2 and water – and as a result no solvents, phthalates or petroleum by-products get into your air.

At launch Orbella comes in three flavors:

  • Patchouli, an earthy scent enjoyed throughout the centuries. 
  • Linalool, a fresh and floral scent. 
  • Geraniol, a rose-like scent found in natural mosquito repellants* 

This is still very much version one, you can smell the moss but it's not strong enough to fill a room yet - that will take more R&D. The patchouli and linalool flavors are much stronger than the geraniol so we recommend those. 

We've put a lot of thought and effort into the packaging to create the lightest environmental footprint we can. To protect the glass orb during shipping we are using a new mushroom based packaging made by Ecovative. This packaging is grown in a mold from waste cellulose materials (eg woodchips or leaf mulch) and when you are done with it you can compost it, leaving zero waste.

Ecovative Mushroom Packaging
Ecovative Mushroom Packaging

 Orbella Ships in a kit to protect during shipping, once you get it you follow these setup instructions:

 

How to redeem your credit

You can order Orbella by following this link: https://orbellamoss.com/products/orbella-fragrant-moss

Once you have selected a flavor you can apply one of the following discount codes where the number is based on the level you backed the project at:

  • GLOWINGPLANT40 - for $40 tier of Glowing Plant seeds
  • GLOWINGPLANT65 - for $65 tier of recycled vase or rootcup
  • GLOWINGPLANT80 - for $80 tier of minimalist vase
  • GLOWINGPLANT90 - for $90 tier of the book
  • GLOWINGPLANT120 - for $120 tier for early bird glowing plant + rose
  • GLOWINGPLANT150 - for $150 tier for plant and rose
  • GLOWINGPLANT250 - for $250 tier for the maker-kit

You must use the email address on file with Kickstarter for the discount codes to work. If you have any issues with this working please send me a message through the Kickstarter messaging platform.

We will ship out the moss in the order in which we receive orders. We are ramping up product but currently only have 400 units to ship before the end of August.

If you have enjoyed the educational nature of our updates you can opt into continuing to receive our newsletter when you order your Orbella.

Celebrate Orbella Launch

To celebrate this milestone we are hosting an event in San Francisco on August 3rd and we'd love you to come and celebrate with us. As well as showcasing Orbella we plan to have an exhibit of some of the other interesting consumer biodesign products being developed. You can find more information here: https://www.facebook.com/events/1372228082897375/

Orbella Packaging
Orbella Packaging

 

Thanks again for your patience getting here. I'm excited to see what you think of it!

Antony

* We have not tested Orbella Geraniol as a mosquito repellent. More information on geraniol's mosquito repelling properties can be found here.

Stopping work on the Glowing Plant

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Backers,

We're sorry to say that we have reached a significant transition point. We were planning to start shipping the fragrant moss two weeks ago, but this has been delayed due to contamination in our production strain. As you know from previous updates our plan was to use the revenues from the moss to fund the ongoing glowing plant research, so the delay has caused financial stresses. As a consequence we’ve had to reduce the size of the team to ensure we have enough financial runway to ship the moss, and this has meant stopping the work we were doing on higher plants to focus on the moss.  

We know this is a disappointing outcome for everyone, and nobody feels this more than we do after putting everything we have into the project over the last four years. We’ve done everything we could to keep this research going but it doesn’t look like that was enough.  

As we indicated in our last update we are working on glowing moss, and hope to keep that work going. The priority now though is shipping our fragrant moss as we won’t be able to deliver glowing moss without delivering that first and we have a responsibility to our equity investors to bring revenue in as soon as possible.

Fragrant moss  

With regards the fragrant moss, the delay was caused by contamination of the moss we have in production. Somehow, for reasons we haven't been able to ascertain, some cells still have a herbicide resistance gene which means we can't ship this batch. We expect to fix this issue but it’s going to take time to regrow the moss back to full volume once we do.  

We have finalised the design of the moss, we’ll be shipping it with a circular glass terrarium and growing it on white rocks. We are branding it ‘Orbella’ and it looks like this:  

Orbella Fragrant Moss
Orbella Fragrant Moss

We have also designed the packaging for the moss. We’ve managed to make virtually all the packaging from compostable materials, so it will have zero waste, including protection for the glass provided by a mushroom grown material in partnership with Ecovative, it looks like this (we haven't printed the boxes yet): 

Orbella Moss Packaging
Orbella Moss Packaging

Since discovering the contamination issue we've also been working to improve the growth rate of the moss so that we can scale production faster than we did before. This has meant moving to a liquid growth system as shown here:

Prototype moss liquid culture
Prototype moss liquid culture

This system is working well, and we've gone from the moss biomass tripling in two weeks to a 5x growth in one week - an 8 fold improvement in growth rate. Our revised shipping date is now July.

FAQ:  

What about refunds?  

As we’ve indicated in a previous update we have spent way more than the kickstarter funds on the project so we cannot offer refunds. Instead we'll offer credits of your kickstarter pledge towards buying the moss and planter once those ship.

What about physical rewards, eg vases?  

Those are mostly in storage in our basement, however we don’t have the funds for shipping and fulfilment.  We hope to ship them once we have revenues from the moss sales.

What about the book?  

This might still happen, depending on finding time beyond the work on the moss, but if it happens it will be more focused on the human elements behind the project, going through YC as one of their first biotechs and the challenges of failing so publicly. The priority for now though is shipping the moss.  

What is the status of the maker kit?  

This is still shipping in beta. Nobody has managed to successfully transform the plant at home with it yet however so we’ve still been making changes to the kit design. We will ship this to those interested in trying to debug it at home. If you want to try the kit, the first step is to get a permit from USDA which you can do by following the instructions in this file: Dropbox Link. 

What are next steps for the project?  

We still believe in the long-term potential of this project. The imperative to move the world towards a lower carbon and non-renewable resource footprint is as strong as ever. Synthetic biology and it's ability to enhance natural, renewable process remains a strong candidate to deliver that cleaner future. Unfortunately, we almost certainly launched this project many years too early with too many technical issues to overcome. Progress in the field over the last four years has been substantial: DNA synthesis has fallen from around $0.25 to $0.08 a base pair, cloud based robotic labs have become available and CRISPR has made fine edits routinely possible (which among other things will mean accelerated understanding of gene function). 

One of the most frustrating things about where this has ended up is that we never got to get all six genes into a plant. It's still unclear whether this is caused by toxicity of the genes or challenges with getting such a large construct inserted correctly. Our primary hypothesis is the latter so we are hoping someone else would be interested in carrying on the attempts to insert the genes. If you are interested in taking the DNA we've made and working on transformations yourself please get in touch at antony@glowingplant.com so we can send it to you - hopefully someone else can build on what we've done and figure out the remaining steps.

Conclusion

This has been an undeniably challenging journey, filled with the kind of highs and lows that you only get when you have the privilege to work on something that's impactful and meaningful. It wouldn't have been possible without your tremendous support and enthusiasm funding this campaign.  Thank you also for the many messages of support - both online and privately - as we've gone through this process, that support kept us going despite the challenges we faced. I'm sorry that we've let you down on delivering the Glowing Plant. I hope though that despite that failure the project can still leave a positive legacy in inspiring people to learn more about synthetic biology and its benefits - and hopefully one day someone does finally make a Glowing Plant.

Yours,

The Glowing Plant Team

Glowing Plant End of Year update

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Backers,

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.

A flat of plants with Lux CDE
A flat of plants with Lux CDE

 

Maturing plants with Lux ABCDEG
Maturing plants with Lux ABCDEG

 

More plants with the full construct
More plants with the full construct

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.

Genotyping

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:

How primers link to DNA
How primers link to DNA

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.

Glowing Moss

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:

Example moss graffiti
Example moss graffiti

Fragrant Moss

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.

Fragrant Moss Production Setup
Fragrant Moss Production Setup

 

Closer view of Fragrant Moss Production
Closer view of Fragrant Moss Production

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:

Physcomitrella patens
Physcomitrella patens

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:

Fragrant Moss At Parsons School of Design
Fragrant Moss At Parsons School of Design

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:

Glass terrarium with freshly homogenized moss
Glass terrarium with freshly homogenized moss

 

Star shaped moss in glass petri dish
Star shaped moss in glass petri dish

 

Egg terrarium with multi-color stones
Egg terrarium with multi-color stones

 

Sphere with white stones, we think it would look better with larger stones
Sphere with white stones, we think it would look better with larger stones

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.

Conclusion

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.

Regards,

Antony & the glowing plant team

Glowing Plant September backer update

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Hello Backers,

In this months update we've got some information on the progress with the autoluminescent glowing plant, some information on the start of our attempts to make glowing moss, an educational video explaining homologous recombination, update on the fragrant moss, and a promo code for synbiobeta where we will making our first public demonstration of the fragrant moss.

Glowing Plant 

In continuation of the effort, we are still in the process of screening and regenerating tobacco tissue bombarded with our full construct. We are still lacking a plant with all six genes but now have many lines with three to five genes. As we explained in our last update, we are going to compliment these lines with a line that contains the missing genes. All of our lines are missing luxC thus, we started work on creating a transgenic tobacco line by inserting a DNA piece containing the luxC cassette. Bombardments have been initiated and screening and regeneration of these lines are in progress. 

Plants derived from this screen will be crossed with the previous lines to complement the missing genes. This seems like an extra step, however, remember we are using a new selectable marker cassette which does not need to be crossed out: Overall, the number of generations the plants have to go through to get the final shipable seeds remain the same.  

Glowing moss

Probably, you all are aware of our Fragrant Moss and that we've started work on a Glowing Moss in case the tobacco plant doesn't work out as we want it to.  Our first attempt is a basic demonstration using the Firefly luciferase cassette, as we did previously in tobocco. In parallel to the proof of principle, we are working on creating different versions of constructs containing the six autoluminescence genes. Some of you might wonder why we should make new constructs when we have the working one for plants. There are couple of reasons:

  • First of all, the dicot promoters do not perform well in moss. For our autoluminescent plant, we are using endogenous promoters from Arabidopsis. However, researchers have shown that the performance of these promoters are minimal in moss. In order to achieve a strong constitutive expression of the pathway, we need to use strong promoters from monocots such as corn or rice that are known to perform well in moss. 
  • Another reason is based on the fact that moss transformation uses a different mechanism from plants. While we force the insertion of exogenous DNA into plants using biolistic methods, moss uses a system called homologous recombination. Detailed explanation by James can be found in this video below. As we only have a couple of known strong promoters in moss, the way to create a six gene construct requires repetitive use of these promoters. Currently, we are not sure how the repetitive elements will affect the transformation process. In order to overcome any difficulties the repeats provide, we are in the process of designing various ways of introducing the DNA pieces into the moss genome. More information will follow in future updates.

What is homologous recombination?

One of the unique features of the strain of moss we are engineering is that it can do something called homologous recombination. This is a specific mechanism for how DNA interacts within the cell, typically found in prokaryotic cells rather than eukaryotic cells, which is explained by James in this video (sorry for sound quality, use the subtitles if you can't hear properly):

Update on fragrant moss

We are ramping up production of the Hygromycin resistance-free fragrant moss line which is the version we plan to sell and distribute to keep funding the glowing plant research. After some initial problems with a fungus infection in August we've cracked the process for scaling up and manufacturing larger amounts of the moss. Currently we can double or triple the amount of moss we have every two weeks. 

We are also testing various growth conditions for moss that will enable it to grow faster, and experimenting with ways to track growth of the moss on agar plates. For instance, we plan to buy a tissue homogenizer that will allow us to more efficiently break apart clumps of moss to spread on several different plates, enabling faster propagation and a shorter doubling time.

Looking forwards, we have a list of ten candidate scents to engineer in moss in the future. We will also be starting to sell and distribute the moss once we have enough to ship in volume. We are still finalizing the plan for exactly how we are going to do that.

Synbiobeta + demo of Fragrant Moss

We are excited to announce that we will be demoing the fragrant moss at the Synbiobeta conference in San Francisco on October 4th and 5th. It was at Synbiobeta last year that we met our Danish collaborators on the moss, so it's appropriate that it's the first location for a public demo. Glowing Plant backers can receive 20% off the registration price if you book before the end of September: just use this link: synbiobeta.com/conferences/san-francisco-2016/ and promo code SBBPARTNER20.

Hope to meet you at Synbiobeta, otherwise we'll be in touch with the next update.

The Glowing Plant team