Depositing plasmids to Addgene (MTA issue)

[Lucelenie]

Depositing plasmids to Addgene (MTA issue)

[Lucelenie]

"The next step is to provide the plasmid information for the vectors you wish to deposit. As this can be time-consuming, I would advise starting with your most-requested plasmids and working your way through in batches. After you have deposited the first 10-20 it's easy to add more. There are a couple of ways to provide us the cloning information for your plasmids: you can fill out our online system and add individual plasmids by publication here:

http://www.addgene.org/depositing/start-deposit/

Or you can fill out our pdf submission form for each plasmid, or more conveniently for larger numbers of plasmids, our spreadsheet template (I have attached these). Are your plasmids a set that need to be used together or are they mostly for individual use?

If you use the spreadsheet or pdf, you can email me the associated maps and sequence files for your plasmids."

[jchodera]

Awesome!

The MacroLab has cloned all the genes into their own plasmid (expression vector) with N-terminal modifications (His6-TEVsite-), so these would be considered new, modified plasmids that are our IP (since we contracted the MacroLab to do the work).

I imagine people may want to request the plasmids as either a set or for individual use. I'm not sure what will be most popular.

One thing to keep in mind is that the kinase plasmids are intended to be coexpressed with either YopH phosphatase for Tyr kinases or lambda phosphatase for Ser/Thr kinases---I don't even see this mentioned in the current manuscript draft. I believe the MacroLab prepared special versions of these plasmids (though I am not 100% sure), and we will want to make sure these are available through AddGene as well. You will likely need to check with Chris Jeans from the MacroLab.

Since the plasmid expression vectors are all the same, and we should have the full sequences of the inserts from the validation sequencing, we should be able to generate a spreadsheet for all of the plasmids and send that all at once to get the whole set in AddGene.

When we reach out to Chris Jeans of the MacroLab, we should also offer them coauthorship of the paper if they want it, since they did all of the experimental work here. They might decline, but it would certainly be appropriate to offer! If they decline, we should ask how they would like to be acknowledged in the paper.

[danielparton]

Good point - I forgot to mention the part about YopH and lambda
phosphatases. Looks like it has been added in now (thanks!)

On Wed, Jan 27, 2016 at 4:34 PM, John Chodera [email protected]
wrote:

Awesome!

The MacroLab has cloned all the genes into their own plasmid (expression
vector) with N-terminal modifications (His6-TEVsite-), so these would be
considered new, modified plasmids that are our IP (since we contracted the
MacroLab to do the work).

I imagine people may want to request the plasmids as either a set or for
individual use. I'm not sure what will be most popular.

One thing to keep in mind is that the kinase plasmids are intended to be
coexpressed with either YopH phosphatase for Tyr kinases or lambda
phosphatase for Ser/Thr kinases---I don't even see this mentioned in the
current manuscript draft. I believe the MacroLab prepared special versions
of these plasmids (though I am not 100% sure), and we will want to make
sure these are available through AddGene as well. You will likely need to
check with Chris Jeans from the MacroLab.

Since the plasmid expression vectors are all the same, and we should have
the full sequences of the inserts from the validation sequencing, we should
be able to generate a spreadsheet for all of the plasmids and send that all
at once to get the whole set in AddGene.

When we reach out to Chris Jeans of the MacroLab, we should also offer
them coauthorship of the paper if they want it, since they did all of the
experimental work here. They might decline, but it would certainly be
appropriate to offer! If they decline, we should ask how they would like to
be acknowledged in the paper.

—
Reply to this email directly or view it on GitHub
#2 (comment)
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[jchodera]

I think we are still missing a good deal of information necessary to replicate things, but we can update the bioRxiv version soon. Submitted it last night so I can get a doi in time for submitting the R01.

[Lucelenie]

I created and sent Addgene the list of the kinase and phosphatase plasmids that we are going to deposit.

[jchodera]

Just wanted to check to make sure you also sent the lambda and truncated YopH164 plasmids as part of this set.

It's critical that they be included, since they are essential for expressing these kinases.

[Lucelenie]

@jchodera : Both phosphatase plasmids were added to the list. I still haven't sent any plasmids, just their information because I am waiting on the Addgene representative to review this information.

[Lucelenie]

Some questions that we got from Addgene:

-Would you like the gene insert to be listed as it is in the plasmid name (ie MK14 for MK14_HUMAN_D0)?
-Would you expect scientists to request all of these plasmids together as a set? If so we can package them together as a kit (see examples here: https://www.addgene.org/kits/). If not we will just make them available as individual plasmids (most common option).

[sonyahanson]

My opinions on these questions:

• I think using e.g. MK14 as the plasmid name should be fine. Did Addgene suggest any alternatives? 'MK14 - 2BT10'?
• I would think a kit could be nice (though all human kinases are already available in a kit on addgene: https://www.addgene.org/human-kinase/ ), but we wouldn't to restrict people to that. I guess if I had to choose one it would be that they would just to be available individually.

[sonyahanson]

Also, are we supplying Addgene with the YopH and Lambda phosphatase plasmids?

[jchodera]

Would we want the whole 96-kinase set or the subset that expresses well in bacteria to be available as a kit?

Also, are we supplying Addgene with the YopH and Lambda phosphatase plasmids?

We must. Otherwise this set isn't useful. Those should also be part of this set.

[jchodera]

though all human kinases are already available in a kit on addgene

That's very different from the "set of human kinase domains that express well in bacteria", which is the main point of our paper

[jchodera]

-Would you like the gene insert to be listed as it is in the plasmid name (ie MK14 for MK14_HUMAN_D0)?

We should probably put a bit of thought into this. We want it to be obvious what this is and we want these to turn up in searches.

Information to convey (in order of importance):

• gene (e.g. MK14)
• organism (e.g. HUMAN)
• this construct is the kinase domain
• the vector and promoter?

[sonyahanson]

Oh, this would be how it would be searched for? That's a different story.

[jchodera]

I'm not entirely clear on this. Probably good to poke around at other collections to get some idea as to how they do this?

[Lucelenie]

We are sending the phosphatase plasmids. To clarify, I sent Addgene a list with the kinase plasmids and both phosphatase plasmids.

[Lucelenie]

They will send us the materials to ship the plasmids (tubes, barcoded labels for the sample tubes, along with a reply-paid envelope to post them back) after we decide on these issues:

1. Plasmid name format
2. Will the plasmids be part of a set/kit or no?

[sonyahanson]

Does the plasmid name effect the process of searching for it or can we add other tags?

@jchodera Do you have strong opinions on the kit vs no kit? My instinct is no kit.

[Lucelenie]

Plasmids can be searched by Principal Investigator, Gene, Fusion Protein/ Tags, Article, Backbone, Expression model organisms, Experimental use, Plasmid type, Species of the Insert, Selectable Marker and Resistance. https://www.addgene.org/search/advanced/?q=

[Lucelenie]

Here is the current spreadsheet that Addgene has for these plasmids.
Addgene_Batch_Kinome_Expression_Plasmids(02_18_16).zip

[jchodera]

Can you guys decide the best choice for (1)?

For (2), I think it would be great to have the option for someone to request them in kit form. Ideally, there would be two possibilities:

1. Phosphatases + all kinases that expressed well
2. Phosphatases + all kinase constructs we engineered

[jchodera]

Some thoughts:

• The spreadsheet column "Purpose" says "Bacterial protein expression. Co-expression with Lambda Phosphatase to enhance expression." for kinases and "Co-expression with human XXX kinase plasmids to enhance bacterial kinase expression" for phosphatases. Shouldn't this be "Intended for co-expression with Lambda phosphatase that accompanies this set to enhance bacterial kinase expression" or something?
• For "Growth Strain", is "XL1 Blue" intended to be the strain in which the plasmids were grown up for glycerol stocks, or the strain we suggest to use for expression ("ROSETTA2")?
• Can we fill in the "Sequence" section with the pyrosequenced or intended sequence?
• Maybe we can fill in the "Gene or insert name" with the Uniprot name (e.g. MK14_HUMAN, without the _D0 suffix)?
• Should we attach the biorXiv DOI to one of these fields?
• Anything else we should fill in?

[Lucelenie]

• The "Growth Strain" field is for an "E. coli strain that Addgene can use to distribute the plasmids."
• I fixed the "Gene or insert name" to the MK14_HUMAN format.
• On the "Comments" field, I added "This plasmid was generated as part of an open library of human kinase domain constructs for automated bacterial expression. The work can be found here doi: http://dx.doi.org/10.1101/038711"
• I'll work on adding the sequences and modifying the "Purpose" field now.

[jchodera]

Awesome!

[Lucelenie]

Most recent version of the Addgene submission spreadsheet. They don't require to fill all the fields; right now the information required for submission is included in this version.

Addgene_Batch_Kinome_Expression_Plasmids(04_14_16).zip

[Lucelenie]

@jchodera @sonyahanson Please confirm that this is the plasmid order that we will submit to Addgene.

Addgene_Batch_Kinome_Expression_Plasmids(052316) (1).xls.zip

[jchodera]

This looks really good to me, though it doesn't seem to mention that the inserts are just a single kinase domain. Is there somewhere we should mention that for the *_D0 plasmids?

After we give it the thumbs-up, we should run it by Chris Jeans before sending it to AddGene.

[Lucelenie]

From John's visit to Transcriptic (recording it here for future reference): "AddGene deposited plasmids default to not being available to industry (like Transcriptic). We need to make sure that we check the box that allows our kinase plasmids to be open to anyone when depositing in AddGene"

[Lucelenie]

Addgene received the plasmids today and they can be found here: https://www.addgene.org/depositing/73070/