Plasmids and Recombinant DNA Technology

[@dayhdez]

Wonderful explanations. It is unbelievable how young you are and how well you know what you teach, apart from having excellent teaching skills. The world would be so different if teachers like you were teaching in our classrooms. Thanks for dedicating your life to this, thank you for helping us see things in science clearer, thank you !

[@AKLECTURES]

+dayhdez Thank you for those kind words! greatly appreciate that :)

[@saltyburger5910]

dayhdez he made a mistake at 8:39 btw.

[@nilukiperera306]

dayhdez I agree. Thank you so much!

[@TheDuvee6]

Young??!?!?! He's well in his 50s

[@intanaprilianihasan3424]

@@AKLECTURES nol

[@sp-gy7dj]

I learnt more in these 14 minutes than 2 weeks of lessons, wow thank you

[@AKLECTURES]

+Simona Peneva you're welcome! :)

[@classylilashley]

Your videos are THE most informative & clearly explained biology/biochem videos I've ever come across! Thank you so much!

[@egberuarelydia1312]

your videos lectures simply second to none on the internet.Always well explained.Thanks for all your videos

[@phantom1014]

You're a very good teacher. I take my hat off to you sir, and believe me when I say that I am extremely critical.. You deserve acknowledgment.

[@unepetitecreatrice5190]

I'm french and you really did help me with my homeworks. Your explanations are really easy to understand and it's clear enough in just one sentence. You saved me ! Thanks !

[@janefrancesladyf]

I must say a big thank you. you've helped through med school, with your lectures that are so easy to understand. i can literally listen to you and enter into the hall with pride and the results are always very fruitful. thank you so much for your lectures. it has helped me a lot.

[@miatoxic7263]

Do you know how much I love you, everytime I have a Bio test I come to you, and once the video is done, I understand that topic like the back of my hand. Thanks so much :)

[@berfindogan6167]

Even though I am only in the 4. min. of the video, you explain it in a much more understandable and fluent way than my professor. thank you so much

[@thomaskoh3289]

Your videos are always of such high quality and grade it amazes me how knowledgeable you are! What is your occupation and job? I'm dying to know. Are you a teacher? Or are you doing this as a hobby. Either way, loving your videos. KEEP IT UP!

[@marissam.6376]

YOU ARE AMAZING!!! Wish I knew about your channel 10 years ago!!

[@narymenemaouche88]

Hat down! U just have this conscience that make you giving all what you have in a very easy way! A pharmacist follower from Algeria... Thank you...

[@jeffgregory2477]

I just want you to know that you're awesome. Every time I happen to stumble upon one of your videos, my comprehension level immediately goes through the roof. Thank you.

[@jennaertman9036]

Very grateful I found this video! Crazy how much I pay in tuition at my college and yet I learned much more from this video than from my professor.

[@Urm0mz]

My professor just pointed at the slides with his mouse and talking. I had no idea what he was talking about! Just a bit of showmanship and passion and it's all making sense

[@Snow4LifeNEver]

Really clear explanations. Constant repetitions makes it really easy to follow and to retain the information. Love this teaching method, keep it up! Was able to watch at 2.3x and the pronunciation was still crystal clear!

[@Hafsa_668]

Thanks AK you are one of best lecturer

[@laurilintott4754]

Maybe you cover this in a future lecture but you have glossed over the main difference between pBR322 and pUC18, and that is how you determine which cells have taken up a recombinant plasmid (as apposed to taking up just the starting plasmid). In pBR322 you must use replica plating to determine which cells have taken up recombinant plasmid vs which cells have taken up the starting plasmid. For example, if you were cloning into the tetracycline gene, you would first select with ampicillin in order to identify which cells took up the pBR322 plasmid. You would then check the ampicillin colonies to determine which ones were no longer tetracycline resistant. Ampicillin resistant and tetracycline sensitive cells will have the recombinant plasmid. If the cells are resistant to both antibiotics, then they took up non recombinant pBR322. With pUC18 you can tell in a single step which colonies took up recombinant plasmid because these cells will be ampicillin resistant but will not turn blue because the inserted sequence has disrupted the B-gal. pUC18 has several other advantages over pBR322 but the ability to screen for recombinant plasmid in one step instead of two is a major advantage.

[@fritzschnitzmueller3768]

This is really great. Precise and on point. great presentation

[@zieqahaziqah154]

I"m so glad I found this channel. thank you so much!!!!!

[@user_25815youtube]

you will be the reason i get my pharmD degree. you are amazing. thank you for all your videos!

[@AKLECTURES]

you're welcome Sarah! best of luck with your studies!

[@mohamedahmed996]

did you get it?

[@Chivende]

Wow ❤❤❤...im ....wow..this is wow .... I'm preparing for end of years this is awesome😊😊😊

[@rajkumar-066]

Too good explanation, I an very thankful to you sir

[@iveeeffy9406]

by far the best video on this material!

[@leonardlarbi6809]

you are a great lecturer,you are making my studies of doctor of medical laboratory science great

[@nefsa84]

I'm confused at 8:40. I thought the original plasmid had resistance to ampiciln AND tetracycline. Why does the modified plasmid only now have the ability to resist tetracycline. I thought it already did...

[@Palamaopa]

You're right. What he's saying is that now it can only resist tetracycline, not ampicillin, because the insert inactivated the ampicillin resistance gene.

[@saltyburger5910]

Nefsa yes you are correct, that folk did committed a mistake. The gene which took the desired dna fragment will actually die when amphicillin is introduced to their medium.

[@lenay3054]

isn't that what he said? I don't get where he made a mistake..

[@CombatHackerX]

This is for the future comrades watching. I too, was confused by this, but we just did not have the full picture about how the process works. Essentially, in most examples, they use one selectable marker(antibiotic resistance section) to simplify things and have the cut site be at the MCS. Anyway, in the video's case, once you have the end product (ampicilin resistance gene inactivated/tetracycline activated) you would introduce it into a bacterial culture. To simplify things, you can assume that initial bacterial culture does not have any plasmids inside at all. Thus, SOME bacterial cells will take up your plasmid with tetracycline resistance while MOST bacterial cells will have NOTHING INSIDE THEM(blanks)//will not have the antibiotic resistance. Therefore, once you introduce the tetracycline, you will kill off the blanks while only keeping the cells that have taken up your plasmid. At least, that is how my cell bio class simplified it. I think Luari's answer tho was more informative/detailed "Maybe you cover this in a future lecture but you have glossed over the main difference between pBR322 and pUC18, and that is how you determine which cells have taken up a recombinant plasmid (as apposed to taking up just the starting plasmid). In pBR322 you must use replica plating to determine which cells have taken up recombinant plasmid vs which cells have taken up the starting plasmid. For example, if you were cloning into the tetracycline gene, you would first select with ampicillin in order to identify which cells took up the pBR322 plasmid. You would then check the ampicillin colonies to determine which ones were no longer tetracycline resistant. Ampicillin resistant and tetracycline sensitive cells will have the recombinant plasmid. If the cells are resistant to both antibiotics, then they took up non recombinant pBR322. With pUC18 you can tell in a single step which colonies took up recombinant plasmid because these cells will be ampicillin resistant but will not turn blue because the inserted sequence has disrupted the B-gal. pUC18 has several other advantages over pBR322 but the ability to screen for recombinant plasmid in one step instead of two is a major advantage."

[@ijjiAllenFalle]

cheers mt

[@yosigilad481]

Wonderful lecture! There is a small mistake I indicated at the end of the lecture (where the inactivating incorporation in the beta galactosidase site is explained). The solution will not turn blue in any case (either the cells successfully uptake the plasmid or not), because the beta galactosidase is inactivated. The only indication will be is if the recombinant DNA was successfully incorporated (no blue color then) or not (then the solution will turn blue).

[@bmrasta5943]

Great source of information! Thanks for taking the time to spread education on recombinant DNA technologies.

[@mominjaved7616]

Maa Shaa Allah.. Allahh give u vvvvvv.good mode ov teaching.. Thumbs up 👍 👍 👍 👍👍 4 ur tchng style

[@GeAsita]

Absolutely clear video of recombinant DNA. Congratulations and thanks a lot for sharing, keep it up!!!!!

[@AKLECTURES]

Gabriel Amodeo Thanks Gabriel! I will :)

[@dyaneribbink5520]

I learn a lot by watching your videos. Thank you very much !

[@AKLECTURES]

+Dyane Ribbink you're welcome Dyane

[@rafaelbezerra9160]

Great explanation, Great teacher skills!! This video was very useful to me. Thanks!!

[@rachelgrace6708]

The best channel to learn !!!!!! Thank you soooo much...

[@zizihassan3437]

my prof just explain the lecture yesterday, i didnt understood completely,now i can understand it even better than my prof 😂😂😂 , thank u thank u thank u u r perfect man god bless u

[@riamirto5002]

You are the best.!!!You do amazing work! Thank you for sharing

[@timsy6869]

Wonderful.Claps Claps Claps. I love the way you explained. Very simply you clear this thing in my head. Thank you. Iam waiting for more vidoes like how to read the DNA digestion, why we do it etc etc.

[@myudadwitamaa2231]

He must be an Italian, just kidding. Your video is every effective, it's really help me a lot. Thanks

[@MrRoperete]

How does this only have 702 likes? THUMBS UP!! This is amazingly useful. Thank you for dedicating your time, hope you get some money from it!

[@1joannabullen]

Thank you so much for your brilliance and excellent teaching when tackling an intricate and puzzling concept. . My university lecturers could learn so much from you. Please continue your wonderful work.

[@keganmengel8215]

Awesome! Thank you

[@righettilea]

Thank you so much! It helped a lot. With your videos I will be now able to understand my classes. Thank you again and keep doing this

[@trusttheprocess08]

you are supper supper. i enjoyed this lectures and took notes like a student mean while am a teacher.

[@ΚαλλίαΣότα]

Such a detailed explanation! At "Recombinant DNA: Genes and Genomes - A Short Course" book about the last section of your video says that the plasmid does not contain the whole beta-galactosidase gene back the part of the gene that produces the enzymes N-terminus (part a). We also have bacteria that contain the part of the gene that produces the other part of the enzyme (part Ω). When a and Ω are together they produce the active form of the enzyme. However, I do not understant why do we make our lives more complicated, but it seems that the first part of the gene is used for the production of a lot of biopharmaceutical products, and my question is why do we use only a part of beta-galactosidase gene and not the whole one? Is it because of the size limits?! Keep spreading your endless knowledge!

[@junczhang]

ive been watching your videos for months. Thanks!!

[@abadinino]

Wow, I found it very helpful and the way you presented it is outstanding. Keep the good work

[@eatandrepeatt]

Now I gain answer of my question topic...... Thank you Sir.......

[@H-Bfit]

If the genes become inactive, thn how the recombinant dna is used for further purposes lets say production of insulin?????

[@geraldzirintunda]

I find this very useful especially for us who missed molecular biology classes

[@samad934]

thank you very much for these lectures. every day i watch these videos. i am a biochemistry student. and these videos help me a lot. again 1000 thanks.

[@uwunayeon9939]

Very helpful! Much needed for my upcoming exam

[@LisaSallaz]

Why am I just finding these lectures? wow love this

[@carolinemangare3071]

Great explanations, much appreciated.

[@righettilea]

Thank you so much! It helped a lot. With your videos I will be now able to understand my classes. Thank you again and keep doing this 😊

[@Curious_Human954]

You are a great teacher!

[@asanda6315]

This was so helpful..your explanations are so clear, Thank you!

[@fariskhiro]

Hello. Nice lecture but I have a question. You said the new plasmid is not resistent to tetracycline,while what you did is inactivate only the ampicillin gene, so I understand that tetracycline resistence is not affected, only the ampicillin resistence, isn' t it?

[@brandonpetrovich2023]

Yeah, I had the same thought as you--that portion of the lecture appears erroneous to me.

[@rachetysambaburajendrapras2715]

Very nicely explained about the r DNA technology.

[@retruthdecliesny4435]

Thanks for this, I learnt a lot, you earned a subscriber

[@samad934]

thank you very much for these lectures. every day i watch these videos. i am a biochemistry student. and these videos help me a lot. again 1000 thanks. :)

[@TendaiBanda-wl2gn]

I like your explanation

[@hailetube6194]

good lecture and explanation thank you very much. It's important lesson in my study from ETHIOPIA.

[@veronicacarvajal4138]

What is the significance of inactivating the green gene and having the cell pick up that plasmid, when both the green gene and blue gene were activated to begin with? Wouldnt a cell want a plasmid that enables resistance to both ampicillin and tetracycline?

[@darkmatter1900]

You're a very talented teacher, thank you! :D

[@morganmiller1282]

Why do we use more than one antibiotic and sometimes even three of them in our plasmid? Wouldn't we only need to put in one resistance gene to see if our plasmid was taken up in a cell?

[@km2052]

awesome as always

[@purnimasaxena7617]

your all lectures are very good ,conceptual and informative which make us fell biology interesting. Sir. i had a doubt.... the plasmid pbr322 which we are using is extracted from E.coli which already has resistance to both ampicillin and tetracycline. After insertional inactivation, ampicillin resistance is no longer and when this is reinserted in the host cells(mostly again E.coli which has dual anti biotics resistance) so how will you differentiate them now?? if you use tetracycline in culture medium then none will die and if you use ampicillin our desired cells will die???

[@bill3430]

Yes I thought that too

[@emilygreen2436]

you're so good AK!

[@kirankavin2826]

crystal clear!! thank you so much ak!!!!!!!!!!

[@ChinchillaHappyLife]

Really good lectures!!!

[@RaMa-ct5qo]

Thank you so much ❤️❤️🌷you are amazing I wish all the best for you 🙏🏻I have an exam and your videos was so helpful

[@georgel8411]

top notch instructor!

[@arungoutham3981]

grt and best lecture..........

[@Ryoma_akaya2184]

It’s so clearly. Thanks a lot.

[@sararizk5502]

You are a saviour!

[@rahafrahal5960]

you are amazing, i like your videos so much

[@mohameda.b6125]

Thanks sir. So informative. Best wishes.

[@veronicacarvajal4138]

Why wouldnt the original cells have the ability to resist tetracycline with the green gene activated, what about inactivation of green gene allows it to be activated?

[@yasiralruwaili4315]

This is just perfect. Thank you a lot.

[@fatouceesay7492]

Thanks for the lecture. How can I get the notes

[@egbejustine]

Does it mean that prior to screening...the host cell (bacterium) that is to be transformed would have it's plasmid rid off...?? Saying this because we know that bacterium cell has plasmid in it. There might be a possibility for a plasmid that is in a bacterium cell may not take up the recombinant DNA but would confer Antibiotic resistance

[@g.sudharsangovindaraj9634]

must check it out!!!!!! explanations are mind blowing and interesting!!!!!!! go AK!!!!!!!!!!

[@bibhadahal7392]

Best explanation ever..thank you..keep uploading :)

[@calebm9000]

Wouldn't it be more efficient to add ampicilin to the solution since that's the gene you inactivated, or do the bacterial plasmids not have resistance for either one?

[@kareshma89]

thanks alot but can you please state the difference in transformation between gram +ve and gram -ve bacteria

[@mohsenteimouri9071]

Hello, You inactivate the ampicillin resistance region. It means if we add ampicillin antibiotic to the colony, the bacteria that take DNA fragments will not survive. However, you mentioned tetracyclin will die the cell. While the tetracyclin regions are intact. Am I right?

[@maryamtahir231]

The recombinant plasmid is inserted back into the same bacteria from which the actual plasmid was taken or is inserted into some other bacterial cell?

[@adilakhalil4183]

this explanation was superb...I got clear idea about vectors. Salute from Pakistan

[@irenevitali6187]

how can we get the little DNA fragment to locate into the plasmid if the restriction enzymes cut the DNA molecole without create a little fragment? I don't know if I express my doubt in a clear way...

[@maryamtahir231]

The recombinant plasmid is inserted back into the same bacteria from the actual plasmid was taken or is inserted into some other bacterial cell?

[@dannyholley]

Superb lecture.

[@AKLECTURES]

+Daniel Holley Thanks Daniel

[@shabdulishinde3685]

I was an idiot in class to gloss over these topics when they were taught. Now I need to go to KZbin videos for help because opening books to learn this puts me straight to sleep.

[@GK-ef1ve]

wish I'd known about this channel earlier

[@haroonsarwarful]

How do you know that the cells that didn't take up the plasmids with the DNA fragment already have the gene for tetracycline resistance proteins?

[@nazmusshalehin2872]

Pbr322 is a plasmid that is resistant to both ampicillin and tetracycline

[@cattycatterson]

How do you distinguish between the transformants and the non transformants?

[@safaamashal7545]

thank you so much ... 😄😄 you helped me alot in understanding molecular biology 😄

[@meamea6540]

[@ehsansalehabadi1500]

Thank you very much for your awesome teaching. May I ask you to teach other parts of genetic engineering, esp genome engineering and integration (such as lambda red, CRISPR, TALEN, Zinc Finger, and MAGE)?

[@CitraTheKrumZ]

dear AK Lectures, I have a question about the plasmid. naturally before it's inserted to desired gene, does pBR322 actively produce both protein for ampicillin and tetracycline? thus when the plasmid take up the inserted gene on PstI site it will knock off the resistance for ampicillin. but how do we isolate the cell that uptake inserted gene from the whole cells when they died in ampicillin solution? thanks

[@inchyokk]

+CitraTheKrumZ great question

[@arnes3418]

+CitraTheKrumZ you dont treat the cells with ampicillin cause all the cells, including the ones who took up the inserted gene, would die. you treat all the cells with tetracycline and only the ones who have taken up the plasmid (and the inserted gene) will survive cause the ones who didnt take up the plasmid dont have the tetracycline resistance and will die.. hope this helps

[@CitraTheKrumZ]

+Arne Scheire thank you for the explanation, however isn't all the plasmid has tetracycline site? Thus both plasmid that took the Inserted gene and the one who doesn't will not die to tetracycline solution.

[@arnes3418]

+CitraTheKrumZ but dont all the plasmids take up the gene?

[@CitraTheKrumZ]

+Arne Scheire no, not all plasmid took up the gene fragment. That's why purification Of cell that contain plasmid+gene fragment is crucial. My question is how to collect cell/bacteria that contain plasmid inserted Pst1 when these cell dies in ampicillin solution, while both plasmid contain Pst1 and plasmid only survives tetracycline?

[@jodscience3741]

Wouldn’t it be easier to use PCR for cloning?

[@fifata24]

wow thank you for explaining everything so damn clear !