July update - 5 out of 6 genes successfully integrated into a plant
In this months backer update we are excited to report that our new selection marker is working and we have regenerated plants which have five of the six genes successfully integrated. We also have a short update on the fragrant moss, including winners of the contest, and are pleased to introduce our newest team member, James, who you can meet below.
Glowing Plant transformations
In our last update, we talked about difficulties we've been having inserting an intact full construct containing all the genes. To overcome the low efficiency problem, which is compounded due to co-bombardment with a selectable marker gene, we have been working on a new selectable marker gene cassette which does not need to be crossed out (more info in last update).
We are pleased to announce that the new selection marker works which will be a big help. This allows us to build a single construct with the six lux genes plus our new selectable marker gene. This increases the efficiency of transformation because any plants which regenerate will include the whole construct rather than just having probability of including the whole construct. The trade-off is that the construct gets even bigger.
We've successfully made this new construct and verified that it glows by transient onion bombardments - which shows that it works even with the gene gun. We have been using this construct since mid-June for our bombardments and just started to get regenerants from the earliest runs. Unfortunately, the first few lines we've analyzed didn't glow, so we followed up with genotyping to verify the insertion of the lux genes.
As shown in the table below, all of the regenerants analyzed had one to five of the six lux genes. The results show that breaking and rearranging of the construct is happening towards the middle of the cassette in Lux C. As the defective site seems to be random, we hope to soon get a plant with all six lux genes with larger number of regenerants.
Despite the fact we haven't quite got an intact cassette yet, the above result is still exciting since every regenerant had at least one lux gene inserted. This verifies that our new selection system is working on tobacco tissue. Another value of this system is, as there is no need to cross out the selectable marker gene cassette, there is no waiting period (for the marker gene to get removed) before crossing. Consequently, the missing genes in one line can be easily complemented by crossing the line with another line that possess those genes. Thus there are two main ways we can proceed to getting all six genes into the plant and we are following both in parallel:
1. The onion results show that some DNA is surviving the bombardment process intact, this means there's a non-zero probability of regenerating a plant with all six genes inserted correctly. Therefore it's a numbers game and we can keep regenerating plants until one has all the genes needed to glow. We don't know what the probability is however so this could take too long to be reasonable (the onion was a transient result so involves way more copies of DNA than a single stable insertion).
2. In parallel we are making a Lux C construct for bombarding independently. We can then cross breed that new line with one of the plants above to get a plant which has all six genes in it. This has a high probability of working, it's just going to take a while to make the new plants and grow them to a level where we can cross breed.
We are in the process of bulking up our first flavor of Fragrant Moss. While we wait for it to grow, we have been testing a number of different materials to grow the moss on. We've narrowed down our testing to two materials: sandstone (which can be 3D printed) and the recycled planter that can be biofabricated.
The moss grows particularly well on the sandstone material. Here are some photos of our moss on sandstone: The left photo shows moss growing on the sandstone after 3 weeks. We transferred moss with tweezers onto a sandstone in a heart shape and it seems to be pretty happy on this material.
Above the heart, there are patches of moss growing, which has been grinded down and freshly painted using cotton tips. The center and the right photo shows grinded moss freshly painted onto the plate - it is very easy to apply in any shape you want and grows up into a nice green moss like you see in the heart:
Winners of Fragrant Moss early access competition
We are pleased to announce the winners of the early access competition, they'll be getting the first shipments of the moss. We'll be in touch with them shortly, thanks to everyone who participated. The winners are:
1. Susan Grossman
2. Dave Marks
3. Lukas Kambic
4. Ben Crawford
Welcome new team member - James
Mary recently decided that she wanted more time to work on her personal DIY Bio projects. This gave us room to hire James Anderson-Furgeson, PhD who recently earned his PhD at University of California, Berkeley, where he studied cell growth and division in Agrobacterium tumefaciens, nature’s genetic engineer and one of the premier tools for genetic modification of plants. Here's a short video with him introducing himself:
James decided to come work with us because he is excited by our ambition to develop diverse new applications for genetic engineering, and by our goal of bringing the benefits of genetic engineering to a larger group of people. He thinks plants hold a lot of promise as (literally) green photosynthetic factories, and is glad to be a part of the project.
Thanks for reading. Don't forget to visit our WeFunder page before August 9th if you are interested in equity investing.
The Glowing Plant Team