Plasmid design - protein tags

No video

Plasmid design - protein tags

Рет қаралды 12,065

Katharine Hubbard

2 жыл бұрын

Video used for teaching on module 500709 Cellular Regulation and Biotechnology at the University of Hull

Пікірлер: 35

@pranavbvn5015 5 ай бұрын

I was confused on how to insert a tag into my plasmids and couldn't find any source that could explain how to do it as good as this one! Thank you very much Katharine for this video! Definitely going to look up other videos to learn molecular biology from your channel!!!

@melanie8388 10 ай бұрын

This is beautifully done, thank you so much for taking the time to explain this and drawing everything out in such fun colors! I'm starting a new job working with proteins (which I haven't done before) and needed this!

@kuukuakyeh7955 Жыл бұрын

This is no sympathy, You are superb! Love your explanation!!!!

@tethpheak6745 4 ай бұрын

Thank you so much for your clear explanation!

@abumohammed9659 Жыл бұрын

Thank you it was very helpful Best wishes

@octaviocastillo7670 11 ай бұрын

Love your videos. One question though. Do you need a linker sequence between two proteins, for example GFP + GOI? Also, would it affect overall protein structure for those sequences in between? (I.e. GFP sequence-4 amino acids coming from a RS or something else-GOI

@EnMaAi1995 3 ай бұрын

Hey! If I am thinking of transcribing my plasmid into an mRNA, do I use the entire gene of interest? Or do I try to find the mRNA sequence and use the cDNA of that instead to put into my plasmid?

@katharinehubbard5043 3 ай бұрын

Good question! Slightly depends on what you are trying to do. If you want to eg produce large amounts of the protein in a heterologous system then you probably want the cDNA to make things more predictable , but if you are interested in the regulation mechanisms you might well want the entire gene

@gregorysmith1134 Жыл бұрын

So what if I want to add a protein to an existing reading frame but don't want it to be a fusion' protein, but a polyprotein that can be cleaved by an encoded endonuclease? Do I just encode a restriction site between the host protein and the new one?

@katharinehubbard5043 Жыл бұрын

Not sure I understand your question - do you want the protein to be cleaved by a protease, or the DNA to be cleaved by a nuclease? Let me know and will hopefully give you an answer!

@gregorysmith1134 Жыл бұрын

@@katharinehubbard5043 I want the protein cleaved by a protease.

@abdulateefsani8500 Жыл бұрын

I think we have something like fusion specific protease,, though when u use a tag called Glutathione transferase, which has protease specific sequence dat connect d protein of interest and the tag..

@user-fd4og4nw2k 8 ай бұрын

nice presentation

@estelleloisel6298 2 жыл бұрын

It was amazing ! Thank you so much 😊 Keep going ! :)

@kokillan1649 Жыл бұрын

Hi. I have a question. I want to add a tag using the PCR primer method. If the primer is very long, can I break the primer into two and do the elongation twice? For example, if the primer (leader sequence, RE, tag, linker, or GOI) is 50 bp long, I cut it in half and use a 25 bp primer twice. The first primer contains a linker and GOI whereas the second primer contains a leader sequence, RE, and tag. Is it possible? Please kindly respond.

@katharinehubbard5043 Жыл бұрын

Technically possible I guess (?), but you'd almost certainly be better cloning into a vector that already contains your tag if you need a primer that long. Hope that helps :)

@alhaeri1 Жыл бұрын

thank you great video

@alejandropikoulasplata5524 Жыл бұрын

Thank you so much :)

@jamalsaiedahmad3779 Жыл бұрын

thank you so much

@jessicajiron5052 2 жыл бұрын

thank you so much!

@abdulateefsani8500 Жыл бұрын

Please how do we add mutation in order to remove d stop codon..?? What enzyme can I used to do dat..??

@katharinehubbard5043 Жыл бұрын

You don't need an enzyme to remove the stop codon. All you need is a modified primer that has the correct sequence to remove the stop codon - if you use this modified primer in your PCR then all copies of DNA that are made will have the mutation. Hope that helps!

@gregorysmith1134 Жыл бұрын

Can you do a video on designing polyproteins, including which protease(s) to use?

@katharinehubbard5043 Жыл бұрын

Afraid I've never designed a polyprotein so not my area of expertise! Good luck with your project

@gregorysmith1134 2 жыл бұрын

It seems there should be something to separate the gene of interest from the tag, so that the resulting protein folds properly and is not denatured. Is that the purpose of a linker? I am the first to admit that at the present time I know just enough to be dangerous.

@Mateo-jc9zg Жыл бұрын

I'm curious about that too, how do we know that adding a tag won't have an effect on protein folding?

@gregorysmith1134 Жыл бұрын

@@Mateo-jc9zgAfter more study, I'm sure maybe the tag will be in the regulatory region and not attached directly to the gene sequence. I was hoping the instructor would respond, too.

@nikolai5012 Жыл бұрын

@@gregorysmith1134 Indeed sometimes the tag does interfere with the proteins tertiary structure. Whether the tag is at the N or C terminus as well as which tag is used needs to be considered accordingly. And I'm not sure what you mean about being dangerous.

@gregorysmith1134 Жыл бұрын

@@nikolai5012 like the person who knows just enough about explosives to blow himself up.

@katharinehubbard5043 Жыл бұрын

Apologies I didn't spot this comment earlier! Yes there is always a risk that adding a tag will disrupt the structure or function of the protein, so needs to be controlled for in experiments. Particularly for membrane proteins which often fail to fold correctly with a tag. For example you could express your protein in a knock-out cell (ie lacking the functional protein) to see if the tagged protein complements it and restores the wild type phenotype. Could also make both an N and C terminal tagged version and see if get same results. Adding a longer linker can sometimes help, and obviously the smaller your tag the less likely it is to cause problems. Hope this helps :)

@amdu581 Жыл бұрын

Dear Katharine first of all thanks alot for the video. Secondly i still have 2 questions: If i am to design a fusion protein between my protein of interest and GFP as my tag, do i have to remove the start codon from the GFP encoding sequence as i removed the stop codon from the GoI or is it not a problem for transcription when it runs into another start codon? Furthermore if i want to create this fusion protein without using a vector that already contains the sequence for GFP can i use PCR priming on the GFP sequence to add restriction sites that exist within a vectors MCS, then clone the GFP into this vector, then use PCR priming on my GoI to add the exact same restriction site at the end of the GoI that i added to the beginning of the GFP-sequence while adding a different more upstream restriction site to the start of the PoI? in my head this would allow me to clone the GFP into the MCS of my vector and then cut right at the beginning of the GFP and add in my GoI there but i am unsure if this is a common way at all or complicated in regards to keeping the reading frame.

@katharinehubbard5043 Жыл бұрын

Good questions! 1) you don't need to remove the start codon - it will just read as a regular AUG = methionine 2) You could PCR clone both the GoI and GFP into a multiple cloning site (MCS) if you wanted to, but is much easier to clone into a vector that already has the GFP in it. For example if your MCS has the sites XhoI-EcoRI-BamHI-SalI you could clone your GoI into XhoI-EcoRI and then GFP into BamHI-SalI (assuming these restriction sites are compatible with your sequences). You'd need to do more checking to make sure you had e.g. the stop codons in the right place and that it genuinely made a fusion protein, so would be easier to clone into a GFP vector where you know that the GFP bit will be fine. Hope that helps

@docmoreau7540 11 ай бұрын

Dear sir or madam, thank you for your question, I had same question about start codon in the middle of fusion proteins and now I have an answer.