stable transformation of chlorella vulgaris

[najib abdellaoui]

hello everyone 

i am currently working on a project for production of vaccine based on stable transformation of chlorella vulgaris. the plasmid size is around 6 Kb and i wanted to transformed into chlorella vulgaris using electroporation to get a stable expression of my protein of interest and also i wanna analyze the site of integration into genome.  we have bio-rad gene pulser II in our lab.

so can someone help me for the design of protocol for the transformation.

thanks in advance.

[Scott]

Hello Najib,

This reference might help you with designing your electroporation experiment. You will need to linearize your vector for stable integration.

https://link.springer.com/article/10.1007/s002990050660

There are many protocols for looking at transgene integration sites, inverse PCR being the most popular. There are even kits but I'm old school.

https://www.ncbi.nlm.nih.gov/pubmed/9893713

https://genome.cshlp.org/content/1/2/129.full.pdf

Compare your integration sequences with the Chlorella vulgaris assembly.

https://www.ncbi.nlm.nih.gov/assembly/GCA_001021125.1/

Hope this helps and all the best with your experiments.

Cheers,

Scott

https://opensciencenet.org

[Skyler Gordon]

Chances are that when you go to isolate the genetic material and look for your insertion site, there will be plenty of remaining vector. I'm not sure what method you were going to use to determine this (qPCR, gel validation, sequencing, ddPCR, etc.) but here is an interesting paper about how Eukaryotic cells retain their vectors for quite a while.

http://www.pnas.org/content/113/26/7177

I hope this helps

-SG

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[Skyler Gordon]

Also, I've been told that maintaining a "pure" culture of algae can be extraordinarily difficult and you may want to include some sort of permanent selection factor (i.e. they need the plasmid to survive so they will continue to retain it over long periods of time).

-SG

[Ravasz]

Hi,

I am actually just starting a project to transfect chlorella too. I also don't have a fixed protocol to perform this, although I have experience with electroporating other cell types, from e-coli to human cells. The main problem with chlorella seems to be that its cells are rather small as far as eukaryotic cells are concerned, and they have a thick cell wall. So first one needs to digest their cell wall away before electroporation. Alternatively a gene gun can also be used with which transfection is possible even without digestion. I understand the latter is the weapon of choice usually when transfecting chlorella.

Finding the genomic integration site will be another problem, but this might be done with a simple PCR and sanger sequencing of the product, using a primer within the plasmid and a bunch of random primers. This may or may not work, if it doesn't then probably some high throughput sequencing will need to be done, which will quickly get rather expensive if you want to test multiple single-cell colonies.

Either way, I'd be interested in staying in touch and developing a good protocol for this as I'm in need of it too.

Cheers,

Mate

[najib abdellaoui]

thanks a lot, the transformation i am planing to do is based on using the flanking regions of nitrate reductase which will cause knockout of nitrate reductase and the selection method i will apply will be use of potassium chlorate. the knockout of nitrate reductase gens will protect the transformed cells against the toxic effect of chlorate. i was planing to used as a selection. method.

[Mac Davis]

I can help what do you need? What's your location? What's the delivery molecule? 

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[Dakota Hamill]

http://www.microsynbiotix.com/

On Thu, Jul 12, 2018 at 11:12 AM, Mac Davis <mac.t...@gmail.com> wrote:

I can help what do you need? What's your location? What's the delivery molecule? 

On Wed, Jul 11, 2018, 5:48 AM najib abdellaoui <naji...@gmail.com> wrote:

hello everyone 

i am currently working on a project for production of vaccine based on stable transformation of chlorella vulgaris. the plasmid size is around 6 Kb and i wanted to transformed into chlorella vulgaris using electroporation to get a stable expression of my protein of interest and also i wanna analyze the site of integration into genome.  we have bio-rad gene pulser II in our lab.

so can someone help me for the design of protocol for the transformation.

thanks in advance.

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[Scott]

You most definitely don't want to do PCR on genomic DNA with random primers! Inverse PCR, as I mentioned earlier, is the way to go. It has been use in all sorts of species from bacteria to mammalian cells. You can even do plasmid rescue if your transgene contains a bacterial origin and selection marker but inverse PCR is the way most people look for integration sites. There is a variant where one ligates a primer-bearing adaptor to the digested genomic DNA followed by PCR with a transgene-based primer. You can get kits for that for the young folks who are flush with money and don't like getting their lab coats dirty!

Cheers,

Scott

https://opensciencenet.org/

On Thursday, July 12, 2018 at 12:02:25 AM UTC-7, Ravasz wrote:

...

Finding the genomic integration site will be another problem, but this might be done with a simple PCR and sanger sequencing of the product, using a primer within the plasmid and a bunch of random primers. This may or may not work, if it doesn't then probably some high throughput sequencing will need to be done, which will quickly get rather expensive if you want to test multiple single-cell colonies

...

[Skyler Gordon]

Inverse PCR is also going to be difficult unless you have some sort of mechanical DNA shearing method. Common enzymatic and chemical shearing will break the DNA down into pieces that are just too small.

I would advise using a splinkerette pcr, or following the protocol that I've attached. Both are simple, rather elegant ways to ensure your PCR products come from the desired location in your genome. 

-SG 

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[Skyler Gordon]

If you really want to shear and circularize and then run pcr, I would suggest getting a Covaris

-SG

[Scott]

Splinkerette pcr is a nice protocol. Haven't tried it myself. I disagree on your assertion that inverse PCR is difficult. I've used it numerous times to identify mouse transgene insertion sites as well as some bacterial transposon integrations. No mechanical shearing needed, only restriction digest. You have to pick the right enzyme(s) depending on your transgene. It has always worked well in my hands.

[Skyler Gordon]

How can you be sure that you're digesting in the right locations if you don't know the location of your insertion site? It seems like it could provide 90% accurate data, but the chance of losing something due to fragment sizes being too large or small is there. 

Maybe you can put me at ease.

-SG

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[Scott]

There are many different variations on the protocol but Wikipedia has a basic overview of the technique. The key is picking enzymes that aren't too rare otherwise your PCR product may be too large. A priori you won't know how far the sites are from the integrant so you may have to try a few different enzymes. 

https://en.wikipedia.org/wiki/Inverse_polymerase_chain_reaction

HTH,

Scott

[Ravasz]

"You most definitely don't want to do PCR on genomic DNA with random primers!"

Oh, you are absolutely right! I haven't thought this through very well have I? It was early morning for me... Also, I did not know about inverse PCRs before, so thanks for the heads up.