Sir, when we use the term "Strong Promoter", In what sense the promoter is said to be strong or weak? I would like to clarify that is it due to the fact that more the sequence similarity of a promoter with the consensus sequences, stronger the promoter is. And moreover if the promoter requires less accessory factors to express the gene, the promoter is said to be strong!!! Are these two reasons correct or is there something more to it???
Ranjit, that is a good question. When we say strong promoter, it means that it leads to larger amounts of mRNA to be produced. All the promoters will require same accessory factors (proteins) for expression, but the sequence of promoter determines how strongly they bind and lead to transcription. A weak promoter will only produce weak binding of RNA polymerase and other proteins, and therefore, less amounts of mRNA.
@@SpartanTutorials ok sir, got the clarification.. Thankyou so much....you are really an enriching source of knowledge...... seeking for more and more such videos in the coming days.
And two more practical based Question, l would like to ask is that after introducing the gene into bacteria, after how much time the proteins start getting synthesized in them? 2) After the Proteins get synthesized in the bacteria, should l purify my protein using affinity column chromatography or immunoprecipitation by applying the antibody against the protein??
Regarding the time it takes for bacteria to start making your proteins - if you use a strong, constitutive (always ON) promoter, then the bacteria will start making your protein immediately, and as they go from lag phase to exponential phase, you should see a huge increase in protein production. Remember, both the amounts of protein and the numbers of bacteria matter. You need lots of bacteria producing your protein, and each should produce it in a high amount (ideally). 2) IP is usually not used for protein purification purposes, it is applied to characterize a protein, e.g. to know its molecular weight, pI, etc. Affinity chromatography is the most commonly used method for protein purification if you are going with a clone of your gene. You can clone it with an N-terminal or C-terminal Histidine/FLAG/myc tag, that should give a very pure preparation of your protein. Other forms of chromatography (e.g. Ion exchange, HIC, Gel Filtration) are also used, but Affinity is the best for recombinant protein production.
I forgot to add - if you use an inducible promoter, i.e. a promoter which can be turned ON by adding a small chemical, e.g. IPTG for a lac promoter - this is actually preferred by some scientists - because they can wait for cells to come to log phase, add IPTG, and boom, your protein starts to get expressed. No need to monitor anything before adding IPTG, just make sure the bacteria are growing happily! :)
Your way of delivering information is really great. Keep sharing your knowledge :)
Thanks for the kind words Pratiksha!
Sir, when we use the term "Strong Promoter", In what sense the promoter is said to be strong or weak?
I would like to clarify that is it due to the fact that more the sequence similarity of a promoter with the consensus sequences, stronger the promoter is.
And moreover if the promoter requires less accessory factors to express the gene, the promoter is said to be strong!!!
Are these two reasons correct or is there something more to it???
Ranjit, that is a good question. When we say strong promoter, it means that it leads to larger amounts of mRNA to be produced. All the promoters will require same accessory factors (proteins) for expression, but the sequence of promoter determines how strongly they bind and lead to transcription. A weak promoter will only produce weak binding of RNA polymerase and other proteins, and therefore, less amounts of mRNA.
@@SpartanTutorials ok sir, got the clarification.. Thankyou so much....you are really an enriching source of knowledge...... seeking for more and more such videos in the coming days.
@@ranjitshaw9110 Most welcome! I'm glad I could be helpful!
And two more practical based Question, l would like to ask is that after introducing the gene into bacteria, after how much time the proteins start getting synthesized in them?
2) After the Proteins get synthesized in the bacteria, should l purify my protein using affinity column chromatography or immunoprecipitation by applying the antibody against the protein??
Regarding the time it takes for bacteria to start making your proteins - if you use a strong, constitutive (always ON) promoter, then the bacteria will start making your protein immediately, and as they go from lag phase to exponential phase, you should see a huge increase in protein production. Remember, both the amounts of protein and the numbers of bacteria matter. You need lots of bacteria producing your protein, and each should produce it in a high amount (ideally).
2) IP is usually not used for protein purification purposes, it is applied to characterize a protein, e.g. to know its molecular weight, pI, etc. Affinity chromatography is the most commonly used method for protein purification if you are going with a clone of your gene. You can clone it with an N-terminal or C-terminal Histidine/FLAG/myc tag, that should give a very pure preparation of your protein. Other forms of chromatography (e.g. Ion exchange, HIC, Gel Filtration) are also used, but Affinity is the best for recombinant protein production.
I forgot to add - if you use an inducible promoter, i.e. a promoter which can be turned ON by adding a small chemical, e.g. IPTG for a lac promoter - this is actually preferred by some scientists - because they can wait for cells to come to log phase, add IPTG, and boom, your protein starts to get expressed. No need to monitor anything before adding IPTG, just make sure the bacteria are growing happily! :)
@@SpartanTutorials Thankyou so much sir again for all these clarifications.
I am obliged to you.
@@SpartanTutorials ok sir.
Thankyou loads for all these valuable informations
@@ranjitshaw9110 My pleasure :)