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!!!
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!
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
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.
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
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?
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!
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..
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.
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 :)
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?
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
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!
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-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.
@@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.
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 :)
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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!!!
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!
This is no sympathy, You are superb! Love your explanation!!!!
Thank you so much for your clear explanation!
Thank you it was very helpful Best wishes
thank you so much!
thank you great video
It was amazing ! Thank you so much 😊 Keep going ! :)
Thank you so much :)
thank you so much
nice presentation
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
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.
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
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.
Can you do a video on designing polyproteins, including which protease(s) to use?
Afraid I've never designed a polyprotein so not my area of expertise! Good luck with your project
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?
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!
@@katharinehubbard5043 I want the protein cleaved by a protease.
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..
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.
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 :)
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?
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
Please how do we add mutation in order to remove d stop codon..??
What enzyme can I used to do dat..??
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!
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.
I'm curious about that too, how do we know that adding a tag won't have an effect on protein folding?
@@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.
@@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.
@@nikolai5012 like the person who knows just enough about explosives to blow himself up.
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 :)
hbb147-honey