The cas 9 and sgrna will go into a plant plasmid
The donor template will be also go in plant plasmid also- pcambia 1201,
Both are going to fix a defective gene in chlorella-microalgae plant cell-
Is it possible that both plasmid can be plant plasmids?
Hi Yaun how are you?
Yes Yaun i dont know if it is scientifically possible. For plant derived microalgae the only plasmid that would be best is a plant plasmid!
1). The sgrna is gonna go in pske401 plant plasmid it already has the cas 9 integreted into it, What exactly is zcas9 Yaun how is different from regular cas 9?
2). The PCAMBIA 1201 plant plasmid is gonna have donor template inserted via restrictrion enzymes?
There both plant plasmids!!
I
I delete my very first 2 answers, becuase I thought you are puting 3 things (Cas9, sgRNA, donor template) directly into a binary vector. But you are doing two binary vectors ("plant plasmids").
Yes, it is possible. But, you did not specify how you going to deliver the vector with 'donor template' into plant genome?
Are you going to do Agroinfiltration (transient assay) for your experiment? To transform both plant vectors ('plasmids') into plant leaves?
Not quiet Yahn. The defective cell is a chlorella microalgae. Its gonna have 2 plasmids inserted to fix the bad gene inside via hdr.
Ok, so look like you are using plant protoplasts for electroporation. In this case, the 'donor template' can be put either in a binary vector (pCambia) or non-plant vector (non-binary vector).
But, one advice, doing such experiment, you might need a lot of plasmid as 'donor template'. And pCambia vector is a low copy number vector, so to produce enough of them for your experiment, you need to do a Midiprep or even a Megaprep to obtain the quantity needed. This can be done by using kits.
However, there is one advantage if you place the 'donor template' into pCambia: you can transform both plant plasmids (pske401 + pCambia) separately into Agrobacteria, and do a quick Agroinfiltration (a tranient oassay) n tobacco leaves to see if your 'plant plasmids' work before you put them into protoplasts.
Hi Gary, I could not find pske401 info online, could you give me its site, so I can take a look what is going on. Thanks.
I got your email. I will also look into your other question.
Do you have the link where you purchased pSKE401, or its instruction sheet??
Its PKSE 401 not PSKE 401 yaun, Sorry! It can be seen here:
https://www.addgene.org/62202/, its the second image with the zcas 9 in it!, how is that different from regular cas 9!
Where did you get this pSKE401? Produced from your previous lab member?
No, its gonna be used as a sample plasmid for now where our guide rna will go under the respective promoter. But it has zcas9 in it...
Ok, I found this paper. zCas9 is described. It is a Zea mays codon-optimized Cas9. The 'regular' Cas9 gene is from bacteria, which might not express well in some plant species. So, people change the codons of some amino acids to make the gene more suitable to express in plants. This is called 'Codon Usage'. And the process is called 'Codon optimization'. The final protein amino acid sequences are the same for the two, but the codons can be different between regular Cas9 and zCas9.
This is the paper I mentioned in the previous answer. See the yellow highlight.
Will that have make any different on microalgae plants cells, so should i stick to regular cas 9?
That is a good question.
I am not sure whether the zCas9 (specify for corn) or the regular Cas9 can express well in your particular microalgae. You can only find out by experimenting them. I have a suggestion: go online and check what Cas9 has been successfully used in microalgae. If the regular Cas9 works, then you probably should stick to the regular one. But since you have already bought the zCas9 vector, why don't you just play both to see what are going to happen....I would do that. Maybe the zCas9 is better, you never know...A lot of great scientific results were discovered this way...
Ok, I just went to the site.
Yes, correct, it is maize-codon optimized Cas9 (zCas9).
Gene/Insert name: zCas9
Species: S. pyogenes
Mutation: maize codon optimized
Promoter: 35S
Tags / Fusion Proteins: •3x FLAG (N terminal on insert) •NLS (N terminal on insert) •NLS (C terminal on insert)
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