Thanks very much for the concise summary! I have got 3 questions: 1.why do we need to include a lacO upstream of the T7 promoter? Won't the downstream one suffice in blocking the transcription? 2. Can we put the de3 cassette into the vector instead? So upon +IPTG into the transformed E. coli, T7 RNA Pol will be synthesized, which can then bind to another T7 promoter that drive the gene of interest (GOI) expression? 3. To confirm, we put the GOI in reverse of the lacZ gene. But when we induce the GOI expression, will the disrupted-and-reversed lacZ gene be transcribed into the same mRNA with the GOI, eventually disrupting the protein sequence? Thanks again!
Beautifully summarized! What is the reason for having Lac operators both on the genomic DNA and in the vector though? Wouldn't it suffice to only have it on the genomic DNA since only T7 RNA polymerase recognizes the promotor in the vector?
The operator is "leaky" it allows some expression at all times otherwise the cell would never import the lactose. By having it both in the genome and on the plasmid one gets much tighter control and lowers the level of expression in the absence in the inducer.
Its normally knocked into the e.coli genome replacing LacZ and under the control of the lac repressor protein. Its insered by prophage (Engineered phage) that has T7 RNA polymerase controlled by Lac regulatory construct.
Norbi Gőcze I was referring to the gene of interest that would be inserted into the MCS in the position of the lacZ gene. If that insertion has not occurred the gene is absent and the lazZ gene is intact. If it is broken by insertion it would prevent the cells from turning blue and so they would be white.
This video is very help full... Thank you so much Dr. David Smith
David, thank you very much, you couldn't have explained it any better or simpler - I use this video as an education to my students :)
This made learning it so much easier! Thank you!! :)
Thank you so much ! I have an exam in a couple of weeks and this video explains so much yet so easy, thanks !
Thx for this good video. I have to study it for my exam in 2 days you helped a lot!
thank you Sir, I learnt the basic expression from your concise video.
Thank you so much for this video. It helped me a lot!!
Great video !
OMG, thank you, this video let me understand finally the topic. c:
It just clear from one video.. Thank you sir
Thanks very much for the concise summary! I have got 3 questions:
1.why do we need to include a lacO upstream of the T7 promoter? Won't the downstream one suffice in blocking the transcription?
2. Can we put the de3 cassette into the vector instead? So upon +IPTG into the transformed E. coli, T7 RNA Pol will be synthesized, which can then bind to another T7 promoter that drive the gene of interest (GOI) expression?
3. To confirm, we put the GOI in reverse of the lacZ gene. But when we induce the GOI expression, will the disrupted-and-reversed lacZ gene be transcribed into the same mRNA with the GOI, eventually disrupting the protein sequence?
Thanks again!
I am interning at a lab right now and found this very helpful, thank you!
Thank u sir, ur explanation was so simple
But why do we need to have Lac operator to be suppressed initially and only then to remove suppressor?
Fire video!!
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Beautifully summarized! What is the reason for having Lac operators both on the genomic DNA and in the vector though? Wouldn't it suffice to only have it on the genomic DNA since only T7 RNA polymerase recognizes the promotor in the vector?
The operator is "leaky" it allows some expression at all times otherwise the cell would never import the lactose. By having it both in the genome and on the plasmid one gets much tighter control and lowers the level of expression in the absence in the inducer.
@@DrDavidSmith Got it. Thank's for answering so quickly!
I really appreciate your video, it clarified a lot! Thank you @DrDavidSmith
Glad it was helpful
this is so good
Very helpful Vidio
Thanks!
I want to know where get the pet 28a vector?Pet28a vector is obtained from E.coli DH5 alpha
Adgene are a not for profit distributor of plasmids. The bacteria a either gifts or can be obtained from chemical suppliers such as GE.
@@DrDavidSmith Thank you for answering the query
Great video
how is the T7 RNA polymerase generated? Thanks.
Its normally knocked into the e.coli genome replacing LacZ and under the control of the lac repressor protein. Its insered by prophage (Engineered phage) that has T7 RNA polymerase controlled by Lac regulatory construct.
Thank you very much!
Hi you haven't explained the directionality of lac z, thanks
What is it you want to know? Its promotor runs in the opposite direction to the T7 promotor
You're the best!
Hi thanks for the explanation.
Do u know where could we get the list of all the different types of vector and bacteria cell ?
blog.addgene.org/plasmids-101-what-is-a-plasmid
@@DrDavidSmith Thanks a lot!
plasmid construction and transfection making the cells and animals cells knock out for specific genes for biological studies
no idea whats happening
If the lacZ is broken the cell turns WHITE!!!!!! You said it wrong
Norbi Gőcze I was referring to the gene of interest that would be inserted into the MCS in the position of the lacZ gene. If that insertion has not occurred the gene is absent and the lazZ gene is intact. If it is broken by insertion it would prevent the cells from turning blue and so they would be white.