Create Plant part collection/package

[nickdelkis]

Add package with parts (CDSs, UTRS, SPs, etc.) for plants

[traci-igem]

I also wanted to point out the Glowing Plant collection from Freegenes that could be easily added to the Distribution.

Link: https://stanford.freegenes.org/products/glowing-plant#genes

[nickdelkis]

The MoClo and GoldenBraid parts collections contains so many characterized parts, so I believe we should start by setting a limit on how many parts per type (promoter, terminator, CDS etc.) we should have and then prioritize according to that.

I will create a directory tomorrow with some basic sequences, so we can start building upon it (and have something to discuss on the #plants kickoff meeting!)

[ReneInckemann]

As we discussed in the meeting, it would be good if we could include vectors for agrobacterium mediated transformation.

[ReneInckemann]

also I think this quite new reporter system for plants, could be very useful for iGEM teams, as it does not need any special equipment: the Ruby reporter https://www.nature.com/articles/s41438-020-00390-1

[nickdelkis]

Ruby is indeed nice, although I think it is patented?

On Thu, 3 Feb 2022, 12:53 am ReneInckemann, @.***> wrote:

also I think this quite new reporter system for plants, could be very useful for iGEM teams, as it does not need any special equipment: the Ruby reporter https://www.nature.com/articles/s41438-020-00390-1

— Reply to this email directly, view it on GitHub https://github.com/iGEM-Engineering/iGEM-distribution/issues/145#issuecomment-1028433328, or unsubscribe https://github.com/notifications/unsubscribe-auth/AML3HYBDP73F5P4WSOHQDXDUZGYXBANCNFSM5LZJWZNQ . You are receiving this because you authored the thread.Message ID: @.***>

[ReneInckemann]

Ruby is indeed nice, although I think it is patented?

the iGEM bielefeld team 2021 already used it, but you might be right that there is a patent

[ReneInckemann]

here is a list of suggestions for plant part we could include for the distribution:

• Binary agrobacterium vector (maybe already containing the selection marker) for Lvl2 assembly

Promoters:

• 35s cmv,
• nos (agrobacterium),
• mas (agrobacterium ),
• rbcs (arabidopsis),
• ubiquitin10 (arabidopsis)

5’UTRs:

• rbcs (arabidopsis)
• tobacco mosaic vírus
• cmv1

CDS:

• GFP
• mcherry (or other red FP)
• Ruby reporter
• Cas9 (optimized with Introns?)

3’UTR:

• nos (agrobacterium)
• 35s (Cmv)
• ocs (agrobacterium)

Tags:

• chloroplast targeting signal
• nuclear targeting signal

Resistance cassettes:

• kanamycin
• bar (bialaphos)

I don´t know if this list is to ambitious or not ambitions enough, but I would like to see a basic set, which is as versatile as possible with very little parts needed. I would collect the sequences for these until our meeting and of course we can discuss what to include and what not.

[nickdelkis]

I think it just right. However, we'd need an intermediate vector alpha/L1 vector to assemble the TUs before going straight to L2. At this stage just one option would be enough I think (?), and it could be expanded later, since it would enable one orientation and one TU per vector.

Also, I'd refrain adding reporter genes like GFP and mCherry here (for now), as they are already elsewhere in the distribution and I'm not sure if automation is set in place to distinguish one "duplicate" per distribution.

For resistance cassettes, I'd add the hph gene for hygromycin resistance (although way more expensive as an antibiotic, but stronk)

For promoters, I'd add an Arabidopsis U6 promoter for gRNA expression.

For transit peptides, I'd add a peroxisome peptide.

I think that's all I'd add without expanding the list too much. I'll create a new branch and collection directory shortly.

[ReneInckemann]

I think the reporter genes for Plants are quite different from the codon usage and the GFP even has some changes compared to the bacterial one. Bacterial fluorescent reporter express really poorly in plants. Therefore I would suggest to at least include a few reporters codon optimized for plants.

Regarding the Lvl1 vectors, I was speculating that these have to be on the plate anyway to allow for modular type IIS assembly. But if not, one would need at least a full set of them to allow for multi gene assembly. Alternativly the loop assembly vectors should also work, but then we would need a "loop compatible" agrobacterium vector.

[ReneInckemann]

The rest of your suggestions sounds perfect to me