Glowing Plant

Updates on the Glowing Plant and synthetic biology news

September update

Here’s Septembers update for you. We’ve got some additional information on the process we are using to generate the full plant, using the gene gun, for version 1.0 which we are planning to ship to you around December. As you may recall from the previous updates, version 1.0 will require the addition of ‘glowing plant fuel’ to boost the luminosity of it. We are also continuing to work on version 2.0 and the rose, and have more updates on that process below.

1. Update on shipping of version 1.0
Here’s a video describing what we need to do in order to get the plants ready for shipping:
https://www.youtube.com/watch?v=XVsczdqDIqQ

You can also see a small Arabidopsis plant regenerating in the following picture. This plant is our control, which we tested by inserting a GFP construct, and is about 3 weeks ahead of our first plant. Hopefully we’ll have a glowing plant like this ready to show you in a few weeks:
2. Update on protein engineering

Our protein engineering process continues to perform well, in September we added another 1x improvement to the luminosity. Here’s the graph of the improvement:

There were a couple of weeks that were flat on improvement, one of these we just didn’t get any hits for colonies that were brighter (statistically this happens from time to time). The second week was a technical failure in the protocol. The third week was very exciting as we found a mutation in Lux G that was right beside an earlier beneficial mutation we found, suggesting this might be a hot spot. We haven’t got the sequencing data back yet for the final hit, so don’t know what kind of mutation led to this improvement.

We’ve got a few next steps for this work stream:

The process is working but is slow and taking up all of Jamey’s time so we want to speed it up through automation. We are now refining the protocol so that we can use a liquid handling robot to do all the work and then we can run it 24/7 which should improve the rate of progress and free Jamey up to work on other tasks
We only select for beneficial mutations which cause an amino acid switch in the protein, these should lead to improved luminosity in the plant but we’ll have to test these to be sure. So the next step is to take the mutations and put them into our plant to demonstrate that it improves the luminosity of the plant.

3. Expression in plants

This was the first month we made a large number of variants of our full gene construct for testing in plants, so it was exciting. Unfortunately we had a lot of problems with our transient experiments this month as the experiments kept being overwhelmed with a fungus which was frustrating. It’s quite hard to see but there are white patches in these wells:

We aren’t sure where it’s coming from, but it could be related to the change of seasons. It took some changes to the protocol but we are now running the experiments under more sterile conditions and they are working again. Here are the results of the transient experiments on Arabidopsis:

The left axis show’s relative units, they can compare within one experiment but not across experiments. The above chart shows 12 different DNA constructs. The big contrast is between constructs 1-6 and 7-12. The difference between the two is codon optimization. Strains 1-6 have wild-type DNA in them for Lux C,D,E & G. Strains 7-12 have Arabidopsis codon optimization. It’s obvious that these results indicate codon optimization works and leads to roughly a 3x improvement in luminosity. We say indicate as it’s still possible there’s something wrong with the CDEG construct that went into all of these so we still have to confirm it’s not that problem.

The other insight we got from this data is that the metabolic reaction for making the light is probably not limited by the luciferase (luxAB). We know this because strains 1-3 and 7-9 have wild type luxAB, and the others have codon optimized luxAB but we don’t see significant difference between these two sets. This is valuable as it tells us to focus on the other genes.

The remaining strains show various other combinations designed to test the impact of adding a YFP gene, which some research papers indicated improved luminosity - our experiments don’t replicate that result.

In addition, we’ve also developing the transient protocols for the Rose. Our rose transient protocol is based on agrobacterium mediated calli. The protocols aren’t perfected yet, as we are getting some variability in results due to the different volume of cells that have been transformed. The exiting news though is that we are seeing quite similar results between the arabidopsis and the rose (these are the same DNA constructs as above). This is good news as if it holds over a broader range of constructs then it means what works in Arabidopsis will transfer over to the glowing rose (and maybe other species?).
Conclusion

Thank’s for bearing through the long update. As usual, if you have any questions please ask in the comments.

Glowing Plant’s growing on the space station!

PBS Newshour - what is biohacking?

I am writing to ask for your support for a field trial that is currently under assessment and public comment with the USDA. The goal is to ensure the scientific evidence is represented and to get a fair balance of comments. As I’m writing this, there are only negative comments, including a 14-page document by GeneWatch, the anti-GM group, which includes a lot of false information. GeneWatch is driving a negative comment ‘blitz’ based on fear of ‘unnatural’ technology and there are no voices representing the science side of the story. Without the voices of other scientists, public comment is skewed entirely toward anti-GM. What concerns me is that beyond a dislike of genetic modifications in the marketplace, anti-GM groups are mobilizing to prevent the scientific investigations themselves. GeneWatch has systematically opposed any genetic technology, including the artemisinin project. But the tide is turning toward science-based policy making and your voice can help.

This is where you can help by posting a quick comment before the deadline of Sept 29: http://www.regulations.gov/#!documentDetail;D=APHIS-2014-0056-0001

Federal Register about field trial: http://www.aphis.usda.gov/brs/fedregister/BRS_20140828b.pdf

Project Background

The project targets the world’s most significant agricultural pest for brassicas (canola, cabbage, broccoli) – the diamondback moth (DBM). This is a collaboration between Cornell University and Oxitec Ltd who developed the genetically engineered (GE) strain of DBM, which is designed to reduce DBM pest populations. We have applied to APHIS to conduct controlled field experiments on this moth strain.

This GE moth was developed using the same principle as the sterile insect technique (SIT), which involves release of radiation-sterilized insects to mate with their wild counterparts, thereby reducing reproduction in the wild population. SIT has been used safely since the1960’s successfully control such pests as the Screw Worm. Check out Wikipedia for the fascinating story http://en.wikipedia.org/wiki/Sterile_insect_technique

A key difference is that the GE technology allows for SIT-type control without the use of radiation that can make the sterile insects less able to mate with native insects in the wild. These GE moths are only able to survive as a colony in the presence of an antidote (tetracycline) that is provided for them in their diet in the lab. Before release, these caterpillars are fed a diet without the antidote, so that only males survive. The male-only release means that they are more eager to find wild females, and without access to the antidote in the lab, they cannot persist in the wild. Even if the antidote was found in the environment (implausible), the persistence of the strain in the field is not possible. These GE moths have a genetic color marker to monitor them in the lab and outdoors, so can easily be distinguished from wild type moths. The marker works because an inserted gene produces Red Fluorescent Protein, so the engineered moth fluoresces red when viewed under a specialized light.

Benefits and Broader Impact

There is a need for new methods of agricultural pest control. Insecticides are the main control method, but these insecticides kill a wide variety of insects and animals and wild moths readily develop resistance to these insecticides, threatening both the environment and brassica production by farmers. Greenhouse trials at Cornell have already shown that GE technology offers an efficient method of controlling DBM and, because the technology is self-limiting, it is designed to be environmentally benign. Brassica production is an important part of US agriculture – just last year the US planted out 1.7 million acres of canola, 130,000 acres of broccoli, 63,000 acres of cabbage, and 36,000 acres of cauliflower, according to the USDA National Agricultural Statistics Service. There is a clear need to evaluate and test new tools for controlling the most significant brassica pest.

The Planned Trial

The trial proposes that engineering male DBM moths be released in field cages first, then in an open field about 12 acres in size, along with planted cabbage, on Cornell University’s New York State Agricultural Experimental Station in Geneva, New York. A study is planned to evaluate the ability of these male GE moths to mate with wild females in these cages, and to test the limits of their ability to survive and disperse in the field. The experiments will provide the necessary information on the biology of these GE male moths in the field to evaluate the strain’s safety and potential for use in pest control.

Safety

Only GE males will be released because they can’t reproduce. When they mate with wild females, the female offspring do not survive without the antidote and so they cannot reproduce either. Our previous field investigations have also shown that DBM can’t overwinter in the harsh climate of upstate NY where the experiments will be conducted. It is also worth noting that the same approach has been reviewed by USDA previously in an Environmental Impact Statement which concluded that it was the environmentally preferable alternative.

Further items you might point to in a comment

the need for scientific field evaluation of new tools
regulatory processes need to be science and evidence based
need for new methods to help reduce dependency on pesticides
new tool against insecticide resistance in this moth which is limiting current control options
this technology is species specific – does not affect non target species such as beneficial insects
integrates with existing farm practices
no apparent safety concerns – non-toxic, non allergenic proteins have been used
self limiting gene technology – it disappears from the environment after releases cease
built-in fluorescent marker for monitoring
related technology deemed safe in OX513A (Aedes aegypti mosquito) in Brazil

If you know of anyone else who may be interested, please feel free to forward this note tand ask them to comment before Sept 29: http://www.regulations.gov/#!documentDetail;D=APHIS-2014-0056-0001

Thanks for reading this long email. Science-based decision-making takes time, but it’s better than blind slogans.

Time lapse photos of glowing bacteria

Monsanto, Under Attack for GMOs, Has a New Defender

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