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00:11 Okay folks, let's go ahead and started. Um So I remember a

00:19 of things do you do this The blackboard quiz opens tomorrow? We're

00:26 gonna cover uh 78 and nine as as we've been through nine today,

00:32 are probably up to congregation. Um you know, so the spot work

00:41 , a couple of those due on or on sunday. Excuse me.

00:45 the cost of schedule opens. Remember example, 3? It opens on

00:51 ? Well I guess technically it's midnight . I guess so. Um

00:56 uh so um remember these chapters have covering 21, Not at all in

01:06 entirety. So remember to, you , a lot of stuff we don't

01:09 about in this chapter, so make that you certainly have the exam review

01:14 to keep you on track. Um so Today we're gonna um continue

01:23 up the last bit of Chapter which again is one of those.

01:26 a lot we talked about there, let's just, I'm gonna rehash some

01:30 the main things um from last So kind of overview. This is

01:36 we're looking at uh examining pro karaoke , how they're structured, how they

01:45 from you carry outs. Um The I guess concept here is um this

01:52 how the genes are struck to um , quote the proteins, proteins do

01:58 most of the work of any Um but that he could be controlled

02:03 well and we'll get more into the aspect, starting next time, uh

02:10 up first with what here's how procurers in terms of their genes and organization

02:15 what have you noticed that operation Uh So um organizing unions of a

02:25 pathway typically under control of one It makes it very efficient to

02:31 It goes to express it at once at once and to and to shut

02:34 down all at once. Um Obviously nature microbes are right there, you

02:43 competing with each other. Environmental changes . Uh So where they can be

02:50 is of course going to be key their survival. And so this is

02:54 one important aspect of that. Um control so there's always going to control

03:01 . Um The Yeah. Okay so operator and um regulatory genes always gonna

03:19 a part of uh and it can things of the operators. You can

03:24 . Activators, transcription, activators, things. We'll talk about this as

03:29 go through this section. Um but obviously a very important aspect of

03:35 Okay, regulations. So we're talking regulations as well. These uh now

03:43 involving of the control of multiple So multiple of these. Okay timothy

03:52 that one does that if they are of a overall um function so to

03:59 . We're talking about nitrogen metabolism that a lot of parts um taking out

04:03 sources um Making things out of nitrogen . These are all involved different operations

04:10 controlling them. Okay. And you coordinate and control these the sigma

04:15 Right? So the super factor is um as well whenever as well.

04:22 what guides the ornate primaries to the . Okay. And then the latitude

04:26 transcribed the opera classmates at the end time. So plasmids are in addition

04:37 the chromosome they replicate on their They have their own origin of

04:44 enables them to do that. They certainly have other multiple genes in

04:50 They in comparison the chromosome On the maybe 1-3 to 45 and jeans but

05:00 can't accept their hands and they have like existence. Um uh as well

05:05 other genes catholic pathways and what have . Okay. Uh they can also

05:11 transferable and we'll get into that Okay. But then also we looked

05:15 this other mode of replication that plasmas do. We're familiar with the bidirectional

05:21 that you know the process you're familiar . But they can also execute this

05:26 circle replication. And so typically this in the context of the congregation as

05:32 see a little bit that copying the the other strand that's displaced can then

05:41 shoveled into the recipient cell and then can make a copy of that.

05:49 now each cell has a copy of plasma. So that's how genes can

05:52 transferred. Okay. And so the so as we get into uh you

06:02 variation. My genetic variation in bacteria archaea. Um they we of course

06:10 all of the things of course we'll mutations that arise at a certain

06:15 But we also have sexual reproduction that us to create genetically different combinations.

06:22 . Creating variations. But bacteria don't . Don't have sexual reproduction.

06:28 So they must rely on other sources variation, rotation of course. But

06:33 as we'll talk about shortly these mechanisms horizontal gene transfer and many of these

06:39 with plasma. Okay. And so inherited plastic means that so cannot express

06:45 genes. Okay. And there may be new genes they have don't have

06:50 . So uh I think the last we mentioned was sigma factors.

06:54 So these are what are transient. buy their part of the memories but

06:59 are not. They can come off on right their role is to uh

07:04 the promoter guide it to that starting so that they can begin transcription.

07:12 , so uh the minus 35 minus refers to that uh promoter region that's

07:18 common in in uh in front of genes. Um we look at some

07:23 of that. But so one thing do want to mention here is because

07:29 talked a little bit about last at end, last time about up and

07:37 Mutations. So um mutations by changing bass or two in the promoter.

07:44 saw either an increase or decrease in . Okay so that brought up the

07:51 of weak versus strong promoter. And some of the mention that here briefly

07:58 so a the the binding of memories promote this is going to give you

08:09 level of expression typically that's what we basal expression. Okay it's it's typically

08:16 enough for the cell to do anything . You don't generate enough protein product

08:21 is meaningful. Okay. Were Um It's we're going to see in

08:27 context of the lactose opera on A little bit of this does produce

08:31 effect. But by and large if cell wants um meaningful protein concentration you

08:38 have to greatly jack this up in of expression. Okay and so it's

08:45 about enhancing this. Okay enhancing that for the memories to to promote.

08:53 one of the things that do that is where we this is where hello

08:57 involves things like control elements, transcription, activators, another transcription factors

09:06 . These come together at the Okay and so uh that now this

09:14 . Mhm. There to that where don't see anything on the promoter.

09:21 This is a site below that has high affinity that preliminary has a very

09:25 affinity for that. So binding to promoter strongly equates to lots of

09:30 Okay so if we can bind the binds the more it will then carry

09:35 to make transcripts. Okay so we doesn't promote that level of binding.

09:42 . And so but not every not operations genius and bacteria or uh in

09:50 of you have a strong promoter in of them. It depends on what

09:52 products are and um what they're used . So you don't necessarily have to

09:59 a strong expression of everything but you have certain things but not everything.

10:04 . But you know from biotechnology standpoint you're if you have a particular micro

10:11 you want to. Yeah it has protein of interest to you. That

10:14 a commercial value to you. And want to on an industrial scale you

10:19 lots of stuff, spoonfuls won't You need bucketfuls. Okay so how

10:23 you how do you make a bacteria that work? And naturally wouldn't do

10:26 . Right? Why Why would it so much energy? You're making so

10:29 more of a program that what it needs. It's insane and it wouldn't

10:33 able to survive so but artificially in lab you can do those things.

10:38 . And so one of the things do is manipulate promoters right? Uh

10:43 find the promoter in front of your of interest pedal with it and see

10:47 you get lots of expression. And then those are in that

10:52 That's that's what you're looking for. the point here is um the difference

10:57 basil and high level expression. It contribution of different factors that enhance

11:03 And so our own needs certainly are complicated in terms of control and uh

11:11 at that basic expression begins with transcription but then gets enhanced by the addition

11:17 other factors that only arise under conditions that gene product is needed. Which

11:24 then in particular factors will come into that um arrives due to the need

11:30 that particular gene expression. Okay and what will enhance. And we're talking

11:35 difference of you know between based on level expression, it can be 1000

11:40 fold difference. Okay so it's it's significant and it's it's also it's not

11:46 an all or nothing thing. Okay a it's a continual. Okay maybe

11:51 don't have all of these here. maybe some of these are not all

11:56 . Well that means you don't get much expression right there all present and

12:00 get lots of expression if they're not at all. Very low expression.

12:04 it's a it's a continual. Okay any questions about that? Yeah.

12:12 um so I think this is kind just basically puts everything together here.

12:18 here is a here is a bacterial . Okay so looking at D.

12:24 . A. And both the scents uh antisense strand. Right so the

12:30 . Right. Plus and minus And then we have two genes in

12:35 example again under control of one Right? That defines the opera and

12:42 um uh we're gonna copy the template . Okay, because remember the template

12:47 copying that right? Gives us a strand in this case are and

12:53 Ok, and that's going to contain coding information so that can be

12:58 So remember it's a it's a polycystic message. Right? It's all it's

13:04 just it's not a here a transcript them be okay, it's all one

13:11 . Okay, continuous place histrionic Okay. But within the within the

13:17 there will be the elements you need proper translation. Right? Start,

13:22 , start stop for each gene transcript then uh um and so then the

13:31 . All right, we're gonna translate . Okay, well it needs also

13:36 particular um punctuation marks. If you . Um It needs rather than binding

13:43 like a like a is a promoter some of these problems and binding site

13:48 about orienting it in front of the transcript or the gene. Okay to

13:55 transcribed. Um So um and then course that brings about the formation of

14:01 obviously. Okay, so that sequence called the Shine Del Garden just for

14:07 two people that discover it. but that's where the riders are going

14:12 . Okay, remember the whole polly rival zone zone play represent information.

14:18 ? This can occur virtually simultaneously transcription their rivals also plop on uh to

14:26 ribosome binding site and then migrates the arrivals binds And then starts translating.

14:32 next one comes in behind it. the next one. The next

14:34 The next one. Alright so you'll a rival games all along the

14:38 Okay. There's lots of protein But again never never forget about you

14:45 element right? Because you can all shut down in a heartbeat.

14:49 All depends on what's going on within around the micro. Okay. Um

14:56 questions about that? Yeah. So wrap this up with a question here

15:05 um then we'll move on to horizontal transfer. Mhm. Okay mm hmm

16:14 hmm. Right. Mhm. Okay uh the operator serves as the site

16:32 the the consensus with D. Rather. Okay um The operator services

16:38 for sigma factor binding. Is that ? Mhm. What services what what

16:49 to the operator regulatory protein. Okay that's fox the opera and possesses multiple

17:01 . V one promoter that defines an . Okay. One promoter multiple

17:07 Uh sexual jeans have a single Code for enzymes of differing metabolic pathways

17:15 that that would be inefficient. Gonna on the same pathway. A single

17:21 transcript will contain information for all the genes of the opera. That's

17:29 Yeah absolutely true. Okay so just it up. Here's a Here's the

17:41 on there's the two structural genes and translated. So it's you know expressing

17:49 contains all the information. Um The factors bind to the shindou garner sequence

17:55 a transcript to initiate transcription. The . Okay so d is the correct

18:02 . Okay. Um Any questions? . Alright. Chapter nine. So

18:13 so what kind of back to I earlier about variation? Right so um

18:22 one oh one right, variation is . Okay. Um If we're all

18:29 identical clones of each other um well be operating in the same way.

18:36 what kind of environmental changes or what you? It would be as one

18:40 entity more or less. Right. by having variation right then you have

18:47 subsets of accumulated genes in in in marriage members of the population that inherently

18:57 possibly that better survival for the Because you know if if things change

19:03 what had different selective pressures occur. chances are greater we have valuation because

19:11 members that have that have the most combination of genes for the particular circumstance

19:15 will survive and those offspring will then and then they will produce another generation

19:20 so on. So variation. Genetic is key. Okay and so as

19:28 , sexual reproduction. Okay that inherently lots of variation. Of course mutation

19:34 well. The bacteria have brilliant Carbs you would think right to

19:40 Um And that certainly works. They very fast. Right? So even

19:45 the colony on the plate there will some variants in there. Okay there

19:50 be a 10 million colonies or something up two million cells. Make up

19:55 colony on the plate, bacteria tend have about a 1% spontaneous mutation rate

20:00 a mistake is made you know every million times is not corrected.

20:06 So uh considering how fast bacteria growth I said they're calling in the plate

20:13 have a few variants of a single . Okay um now the computation is

20:21 thing. Okay that can certainly generate . Right? And so uh those

20:26 can be passed on through vertical gene . So just think of parent and

20:31 . That's vertical gene transfer. That's you inherited your genes right? But

20:36 have this option as well, horizontal transfer. So they can transfer genes

20:41 their neighbors next to them. Okay . And um now we're not talking

20:47 a whole chromosome transfers here. It's segments. So a few

20:54 one or a few can be Okay. But nevertheless it provides

21:01 Okay and so the mechanism will go a conjugation, transformation transaction.

21:09 Each has a characteristic kind of feature it um as well. I'll show

21:15 a brief overview here in a But let's look at this is e

21:20 it represents e coli at different Okay so the big blue blob circle

21:29 represents all of the known E coli we are aware of. Okay.

21:37 is like 11. A little over genes. And we've been um sequencing

21:44 coli genomes for decades so we have lot of information there. So uh

21:51 no one no one E coli will all 10,000 plus genes of course.

21:58 . Um it will have roughly about little less than 5000. Okay,

22:06 5000. Um and so then we down further to the red. Is

22:14 core genome? Right? The core what basically all all the coaches will

22:20 ? Okay, core genes Okay. they all share. Okay. And

22:31 if we look at a couple of coli strains, K 12 was just

22:36 basic harmless lab string that we actually . That lab Over 5 7.

22:42 call the chipotle. E coli. , responsible for foodborne outbreaks related to

22:48 produce. Typically I think it is obviously a pathogen. Right? So

22:53 is the difference in the number of the bulk of those being um involved

23:01 um as violence factors causing enabling to disease. Okay, so um just

23:10 the core gene and flexible gene Okay, core genes are what you

23:15 guess your call them informational molecules like involved in protein synthesis in um DNA

23:25 in like core metabolic pathways like like or respiration, uh amino acid

23:35 These kind of things that are critical survival according to your flexible gene pools

23:41 those that generally don't aren't necessary for survival. Okay. But can be

23:49 in certain cases. Okay. But members of the flexible gene pool,

23:54 jeans are the ones that are typically ? Okay. Um Now when we

23:59 at ecology know the percentages here. , 88% protein coding way. Big

24:09 to reality. 1% of the you know is actually used for protein

24:17 . Bye now. Like 2% like something like that. In that range

24:25 about two middle over 2% of human is used actually code for proteins.

24:32 But much more obviously much more so bacterial genes there's a lot of

24:37 N. A. That we have and another a few carrots as well

24:43 we don't know what it does to lot of a lot of it's for

24:47 three. A lot of it's like sequences uh that are not really sure

24:53 that's for either. But um but it's it's uh surprising how little of

24:59 gm was actually used to coat That probably tells you how much how

25:04 regulation is because a lot of the stuff is for that purpose. Um

25:09 anyway, so in e. Coli is typical numbers for any any bacteria

25:14 any pro cario uh 11% for regulation 20%. So for Tianna Roswell our

25:22 to remember that most genes code for but some the dan product can be

25:28 R. N. A. Not protein but we still call those genes

25:32 then 20% of the genes of the acquired from other microbes. Okay.

25:39 um so how would how do you that out that that's the case.

25:45 . So what's done is something like ? So a parameter that you've been

25:51 for a long time says percent C. Okay. Um if you

25:58 what's his name? Charge off the graphs rule when you studied D.

26:03 . A. Structure A. And G to C. Alright.

26:08 those forces are equal G to And agency that was charged off

26:13 Did that. Right. So he percent G. C. Uh parameter

26:18 later it was used for taxonomic purposes be able to it was found that

26:24 have their own characteristic percent G. . Of the DNA. And chromosome

26:29 or DNA. And they kind of it as a way to for taxonomic

26:34 to examine relationships and things. And what we can do is look

26:40 So this is actually valued for E 50 50.8% easy content. So you

26:46 look on the computer courses works done at all this information H. G

26:51 . S and T. S. then you look to see is there

26:56 continuous stretch of nuclear types that is above or below that percent GC

27:03 Okay. Um and of course you make this determination from just looking at

27:09 bases. Right. So you have type of significant number of nuclear ties

27:14 2500 bases is the average size of gene is about 1000 nucleotides. So

27:21 basis is a pretty good chunk. I? So if within that,

27:25 know, we see a consistent Do you see that significantly higher or

27:31 this case higher, then you might that hey this this this may have

27:35 from something Another organism, especially if those 2500 bases they represent jeans have

27:43 common function. Okay, jeans have common function. Like maybe it's a

27:49 resistance or maybe it's a maybe it's small metabolic pathway. Right? And

27:55 genes are involved in it. And so that's a good evidence that

27:59 that came from something else. Well would it be? Well, you

28:03 say, well, what are the percent gc contents of other bacteria?

28:07 , internal factor erogenous, very close of the coli has does have that

28:13 . So you might suggest so maybe got it from a intro background.

28:16 heck. But it's a but that's you do these kind of things.

28:21 . Um and so this we're gonna in a second about what are called

28:28 genomic genomic islands. Right? So islands and these are typically the segments

28:36 DNA that are transferred. Okay. so this might be I still can't

28:43 island apparently island. Okay, so might be in genomic island. If

28:52 if the genes are part of a pathway. Okay that's what characterizes the

28:56 diamond. It's it's something that's sitting the chromosome that clearly came from a

29:01 species. Okay. Or general. um and they're all involved in the

29:06 function that's pre indicated that was required gene transfer. One of these

29:12 Okay. Um Any questions about Okay. Yeah it's it's really stands

29:23 the percent of guanine and cytosine bases types in the D. N.

29:28 . It's generally used to determine the of it's used it's used to the

29:35 came from that guy charge off. are because we know that A.

29:39 T. Base pair together GNC paper and so he came up with the

29:44 for these things. Black. If if it's 50% 50.54.8% G.

29:50 then obviously it's 40 6.2% 80. ? So when he was still a

29:57 studies of DNA and just doing chemical he's found that these ratios are always

30:01 very consistent. That's what led us believe oh A. T. And

30:05 . C. Base pair together. this this predates Watson and crick by

30:10 years. OK and so that allowed piece of information helped um um Watson

30:18 Crick to figure out their structure But since that time since the 50s it's

30:23 used for a period of time as taxonomic tool. Okay so you would

30:29 general bacteria e coli other similar interests gonna have percent gc contents very similar

30:34 each other if they're closely related. different if they're not so closely

30:39 And that does bear out when you that with you know it's not really

30:45 today as a taxonomic tool. Okay use DNA sequencing but there is some

30:50 when you look at DNA sequencing it's based on DNA sequencing and this

30:53 is some consistency. You have sections to have a different gcc percentage.

30:59 when you. Right so that's so that context or not you're really using

31:04 for taxonomic purposes here. We're using to see is there a dramatic difference

31:08 the sequence of nucleotides? That's where the genome. There we go.

31:14 this is you know the further and can we can identify actual jeans in

31:20 and those genes are appear to be of a common function or something.

31:25 I couldn't believe this may be a island required by one of these mechanisms

31:30 horizontal gene transfer and finding Ireland Mhm. Just that it was it

31:38 inherited by a horizontal gene transfer It wasn't it wasn't part of its

31:44 original lineage. Okay. Uh look fixation. Right that's another one that

31:50 here because national fixation you see across wide range of bacteria Okay from gram

31:56 gram negative for the synthetic hetero trophic happy. And so it actually mocks

32:03 . You know we try to figure sometimes they need just because you see

32:07 that's so widely despaired organisms. But fact that horizontal transfer does occur explains

32:14 . Okay, so it's kind of it's kind of like a patch on

32:19 regina somewhat. Yeah. Blue sweater fantastic. Yeah it's something like

32:28 Sure. But but of course it's beneficial to your it's quite the genes

32:35 can potentially give them an advantage possibly know so and and can potentially transfer

32:44 to another organism. Um Okay so mechanisms so here we're just gonna go

32:52 quick and we'll spend a little more on each joint as we go

32:56 Um So transformation. Uh huh on seemed probably the most simple simplistic process

33:05 of DNA from the environment. And don't don't think in terms of the

33:09 chromosomes are being taken up here. talking about fragments of DNA and the

33:14 you know only has a is not stay in the chromosome right? The

33:17 . And yeah we are going to getting too fragmented DNA. So we're

33:21 about fragments being taken up here but it's a little more complicated than

33:26 But um compared to congregation, congregation two selves. Alright, so intimate

33:34 between two cells. Transfer of uh this involves transfer plasmids here. Okay

33:40 fragments of DNA. So transfer plasmids during congregation. Uh so you're gonna

33:46 a mating pair of donor and recipient will undergo this trans deduction virus C

33:55 transaction you think virus the virus is between the two. So the virus

34:01 um it's due to an error in basically. So the virus packages host

34:07 . N. A. Instead of its own viral D. N.

34:12 . Okay. And um then that caption now contains bacterial D.

34:19 A. To infect another host. . And now that bacterium has acquired

34:25 from that prior host. Okay. that's transaction transposition. So transpose seasons

34:35 widespread among prokaryotes, eukaryotes. We them. Um Typically there they remain

34:44 the organism there in and what they is they jump around the chromosome two

34:49 spots. Okay. But occasionally they exit the host and go to a

34:56 host. If they hitch a ride a plasma for example. Um And

35:02 course we're talking here in the context precarious, the transposon jumps out the

35:06 onto a plasma and the plasma is to congregate and then as for it

35:11 way there's a couple of other mechanisms that's the idea. Okay and among

35:18 it's known that there are a few resistance genes that are passed this

35:23 Okay. I believe in terms of of horizontal gene transfer. Mhm congregation

35:33 deduction. But the more common Okay transposition somewhat less. So although

35:41 does occur obviously. But in terms proportions I'd say congregation transaction are more

35:47 in terms of this horizontal gene Um Alright so speaking of just mentioned

35:56 islands. So again these are elements are grouped in the chromosome. They're

36:04 of a common pathways. So this just showing examples of that. So

36:07 given these names. The names are labels are meant to reflect the types

36:16 types of functions that genes are involved that are part of the island.

36:20 pathogenesis the island relates to disease causing , jeans, symbiosis islands. Genes

36:27 in bringing about the the relationship with host. So think of natural

36:33 Right. Symbiotic nitrogen fixation, bacterium coming together so that the parts involved

36:39 that happening could be a symbiosis island pollack pathway we mentioned before the metabolism

36:46 aromatic compounds that bacteria can do This could be an example of

36:51 Okay these are elements that can be uh required by one of the mechanisms

36:57 horizontal gene transfer. Um But again on the chromosome having these comment

37:05 Okay. Um now. Okay so before we start on the different these

37:12 mechanisms. One last thing to mention recombination, recombination is an important part

37:22 really all four of the mechanisms. because you are um taking D.

37:31 . A. And you're inserting it another piece of D. N.

37:34 . Okay and that involves your Right So if you have D.

37:39 . A. External to a bacterial . For example whether it's a circular

37:44 like a plasma or a fragment then fragment. Well what what can the

37:51 do with that? Okay. The to sell. So you can enter

37:57 conjugation for example. And it can coexist in that fashion. It cannot

38:03 as a plasma inside the cell. no big deal a fragment. That's

38:08 little different. Right? But you're a sense that that's a viral

38:13 Perhaps it's not normal for just a piece of DNA. To be fragments

38:20 DNA to be hanging out in the . That's not normal. And so

38:25 typically get degraded very quickly. Okay a lifetime of something like that is

38:31 short. You can use your right? For example can be used

38:35 food. Um But they're not gonna a linear fragment. Like that's not

38:39 to just sit like that in the . Okay. But what it can

38:43 is it can recombine. Right and part of the chromosome. Right?

38:47 that's that's how it can exist. kind of makes it a permanent part

38:52 the. Okay so um but you we're going to see examples of this

38:58 each each type of mechanism. And so the combination in general is

39:04 to well a number of enzymes but main one record for the combination protein

39:14 . Okay. Um it uh somewhat august to a signal factor. It

39:21 align the the donor fragment with the in areas that are homologous. Right

39:30 the two the two D. A. Is coming together recombining.

39:32 be that different. There has to some level of cosmology because the group

39:38 is all about complementary base pay O. T. G. To

39:42 . Right so there has to be sequences of those that their similarity.

39:48 . And that wreck a helps find and facilitate facilitate that process. So

39:55 do recombine and that donor now becomes of the chromosomes. Okay. And

40:02 course in the process that makes this then a permanent part or semi permanent

40:09 of the genome. Okay. Um of course along the way obviously the

40:15 one receiving that has gotten potentially some jeans. Okay um Again increasing

40:23 Okay so I just wanted to throw in because you're gonna see a combination

40:28 I said in each of the four . Okay. Um Okay so let's

40:34 about transformation first. There's a question transformation. Okay so transformation requires

40:43 Mhm. Yeah mm hmm. Okay let's see. Yeah it's gonna

41:45 DNA fragments in the environment. So cell contact. That's um conjugation.

41:50 intermediates, transaction sex pilots congregation plans going to be congregation uh to the

41:57 . So transpose is is part of transposition. Okay so um so in

42:06 at transformation. Okay uh Uptick of from the environment we have um we're

42:16 get to processes gram positive and gram . Okay and in the gram positive

42:26 involves a phenomenon we've seen before. quorum sensing phenomenon. Okay so remember

42:34 is a cell density dependence. You to get to a certain cell number

42:38 , producing a chemical signal of some but that signal has to accumulate a

42:46 level for them to create the Right? So it depends on a

42:49 of cells coming together. Okay. saw him biofilm formation. So uh

42:55 were positive uh inquires this big multi complexes what they call a transformer zone

43:03 called the trans loca zone because it D. N. A.

43:09 Um It's contingent upon these competence So when you hear the term

43:15 competence goes with congregation, I'm with , I'm sorry but transformation competence and

43:20 go hand in hand. Okay, simply means that when the events have

43:27 place there there now in place in cell to take A D.

43:30 A. You say it's competent? prior to that point it's not but

43:35 it reaches that point we say it's so it's going to take it.

43:39 so there's there's um uh so competence are the little signals that need to

43:45 a threshold for the process to occur a grand positive. Okay um in

43:52 gram negative. Okay they have um that can do this now. Not

43:59 gram positives can do and not all gram negatives can do it. So

44:01 what's called natural transformation. Those are that can naturally do this.

44:06 So by no means is it's something every precarious can do. No not

44:10 a stretch. Right? So for souls that you know so artificial transformation

44:18 obviously a something that humans do to cells pick up DNA. Okay.

44:26 so why why do that? Well has to do with the utility of

44:32 in the lab to help you with genetic recombination, your gene cloning

44:39 Okay um you want you bacteria easy grow and they grow fast and grow

44:45 large numbers. Right? So you that ability. Two replicate your

44:52 Right? So if you have a for example uh that has a gene

44:56 want or of interest and you want of it lots of copies of your

45:00 . You put it in the Let it do the work for

45:03 Okay But E coli for 30 plus has been the workhorse in the lab

45:10 these kinds of things. So we coli as the vehicle for that lot

45:17 of the time. Okay. But coli is not naturally transformed. So

45:21 developed these methods to be able to DNA into it. So that will

45:25 it. Ok that's what artificial transformation . If you took uh we took

45:32 too. I think intro bio I think you did something like

45:35 You had at least you had transformed your D. N. A.

45:38 get competent cells and you had A . N. A. To it

45:41 you made them grow the password for . So what you do is just

45:44 just chemical that can be chemicals, chloride, calcium chloride. Typically a

45:50 shock. You have an ice and shock it and put it back on

45:54 all combined to create pores in the . Such that DNA is taken

46:00 Okay. Uh more recently well last years the corporation has been the way

46:08 do it much more efficient. Um uh that to create spores electric shock

46:15 pours so much easier to do. So regardless so artificial transformation is only

46:22 those that can't do it on the . Okay so with the graham back

46:27 the gram negative over here. Okay that involves a pilot. So remember

46:33 talked about different types of pilots is sex pilots will talk about in in

46:39 . Um If pilots that can be for the twitching motility if you remember

46:44 . Um So pilots is tend to more specialized functions. Right fewer in

46:49 more specialized functions. Okay And this another one. A Type four pilots

46:54 latch on two DNA fragments and bring in to the cell. Okay and

47:02 Demopoulos and nice Iria Our two major types um that are able to do

47:10 um I'm office causes uh one of causes of meningitis. So it's

47:15 Syria causes meningitis um and gonorrhea as . Uh But we're gonna that's all

47:23 gonna say about grandma. So we're focus on gram positives in that

47:27 Let me see here. And so looks more complicated than the gram negative

47:33 involves more parts. Okay, so goal here is to make this

47:38 Okay, that's a complex that will and take up DNA. Okay,

47:45 in order to do that. So , once again, right several times

47:49 I mentioned um you know how much processes take energy, Right DNA replication

47:56 , sensors this as well because we're proteins assembling stuff. Right? That

48:01 energy. So it's not something that going to be, it's going to

48:07 willy nilly. Okay so there has be a right time to do

48:13 Okay and so it's time to the sensing. Okay, so is dependent

48:22 the accumulation of these competence factors. , so they're produced by the self

48:29 if enough cells are present right against enough cells get together you have enough

48:35 accumulated enough confidence factors then you get um you get this happening here.

48:44 so that's a sensor protein that binds factors when they reach a certain

48:50 And then then the end result is activate a sigma factor. Okay And

48:57 that acts on different operations. 1 3 4. And what do we

49:03 that? He called that a Okay. Another example here multiple operations

49:10 control with single element all involved in common phenomenon transformation. Okay um So

49:23 so I guess the question is why density dependent why cell density dependent?

49:31 have to think of of the growth . Right? Time, time and

49:43 number. Right. So we have typical lag lag log stationary.

49:53 And so oops this way. So any given point along this curve

50:01 You have. Okay. The proportion cells that are alive and dead.

50:09 ? Um And as we go along curve we accumulate more dead cells.

50:13 ? We get the ratio of lying dead becomes equal a stationary phase but

50:19 the dead cells that lice and then up their DNA. Those are the

50:24 in the population of DNA fragments like that die that lies and there guts

50:31 spilled, so to speak into the including the DNA they have. So

50:37 guess is that making a cell density process. Right enhances the chances that

50:43 will be D. N. Out there to grab up. Okay

50:47 as more cells to commit that means dead cells are there they lie.

50:50 now you have a source for of to take up. Um That's the

50:55 at least. Okay um And so uh in doing so the I need

51:07 back out of this because doing something . Let me just try this

51:12 Okay all right and back to There we go. Okay so um

51:22 so when the transformation occurs, the . N. A. On the

51:25 external only one strand comes in, is degraded as it becomes as it

51:30 in the cell. And so single comes into the cytoplasm. Remember it

51:36 going to survive, it's gonna need recombine the chromosome, it was going

51:40 become a part of that cell. . Um and so yeah that's that's

51:45 profits for for for gram positive, positive that can do it. This

51:51 generally the process. Um Any questions that. Yeah so yeah I can

52:02 that in the operations uh kind of kind of wondering is that what triggers

52:11 transparency? Oh yeah so that so yeah it's not really shown here but

52:16 products of those operations uh code for different protein parts of that transformer

52:22 Yeah probably as well as well the what's it called? Indo nucleus that

52:28 the one DNA strand. So yeah operas code for different parts of that

52:32 . Yeah. So what's the for purpose of the audience that's tied to

52:39 whole quorum sensing thing. So it's about when you want to explain all

52:44 energy to do this in order to a DNA from the environment is when

52:47 have the greatest chance of finding DNA the environment. And that's gonna generally

52:52 when you have a higher cell density you're gonna have a greater proportion of

52:57 cells at that point. Those are sources for D. N.

53:03 Taking it from the environment. Cell cell dies license DNA. That

53:06 out of the cell. Right? that's that's what the thinking is.

53:15 . Ah The confidence doctors, So the common factors initiate the process

53:22 they won't until you reach a certain density. So it's tied to that

53:28 like biofilm formation dr export is kind showing 80 ft. Yes.

53:40 Right. Yeah. Um So as going on to the next process which

53:52 conjugation? Okay. Um No, a restaurant here. Okay. Um

54:10 uh so the congregation contact between two . Okay. Um it involves a

54:17 of genes that um one of them sex pilots and a number of others

54:24 helped to bring about the uh connects to selves, the copying of the

54:31 and transfer. Okay, so the F factor, fertility factor refers to

54:38 plasma that has those components that enable to be transferred. Okay. And

54:44 are these are what you see here components. Okay. And so these

54:52 the transfer. So remember that you the plan if you say oh that's

54:57 is F. Plus. Okay, means that plant, it has a

55:04 that is transferable but it can have genes on it certainly can be can

55:10 pathway can have anybody. Resistance genes have other stuff on there. The

55:15 Plus designation means it's transferable, can ? And so uh different types of

55:21 involved. So transfer genes they're called included sex, pilots. The relaxes

55:27 the complex is what Bridges The two facilitates the uh the entry of being

55:34 to the recipients. So then there's couple of different story origins of

55:40 The key is where rolling rolling circle is initiated from. Okay. Um

55:48 so we see here in terms of process that a pilot which you see

55:54 the top. Okay. Is and is all about plagiarizing putting the piling

56:00 on it to make it longer or them away. Right. Diploma rise

56:04 make it shorter. Okay, so really alternates between extending okay and contacting

56:10 cell and that's receptor specific and then and then deploy, memorizing it.

56:17 what's happening is as we go down distance between the cells is less and

56:23 . Okay. Until you form that called a conjugation bridge at the

56:28 And so that's all about you know that that pilot is it is rather

56:37 . So if there's two cells are far apart and it stayed that

56:41 The chances of breaking apart are much than if you just bring them together

56:46 this. Okay, so the amount genetic material transfer is totally dependent on

56:53 long they're connected. Okay. The they're connected and more, the more

56:57 that can be shoveled over to Alright. We're transferring plasmid uh you

57:02 , which can vary in size. gonna need to be connected for uh

57:07 number of minutes. Okay, longer you might think. Thanks. And

57:14 so certainly bringing themselves together greatly enhances ability to stay together. Okay,

57:21 Now the process so again the rolling circle replication occurs. Okay. And

57:34 close up here, so you see as the other strands displaced, it's

57:39 shoveled into the recipient cell. So have a donor recipient cell in F

57:44 N. F minus, sell the minus recipient, receives the copy of

57:48 classmate. And then once it does then makes a double stranded copy of

57:53 . Right? And um the and has been converted and if mine itself

58:03 . Okay so um and now the Plus cell is able to transfer that

58:11 with two other mhm. Members of population. Ok. So it's a

58:17 to increase the the spread of the and within the population right to create

58:22 F plus cells, then they can it on and pass on and so

58:25 . Okay, so of course antibiotic can spread it fairly quickly because of

58:31 phenomenon. Okay. Um So now we look at so again back to

58:42 . Okay. Although we're not seeing combination here because the pattern is just

58:46 outside the chromosome in the south. . But this plasma can also be

58:53 with the chromosome. Okay, so of the HFR cell is what we're

58:56 about. So the nature of our requires what to form. Okay,

59:07 let me charge this here. I think I just give the answer

59:13 . I was explaining. Mhm. think I said began with our

59:49 Mhm. Okay, let's see C. F. Factory integrated in

60:18 chromosome. That's correct. Right. I mentioned recombination. So the so

60:23 plans were recombined with the chromosome, it integrates in the chromosome. Um

60:28 again viral intermediate. That's transaction. , A sex pilots. So HFR

60:34 already has the the effect of plasma it. Okay, So £6 isn't

60:42 . Um But it's certainly integrating. is what it's all about.

60:46 so it's an event happening inside the itself. Okay. And so here

60:53 is a pitch. Let me get out of the way. So here

61:00 a picture of that. Okay, we took for instance of a high

61:04 combination strength. So um so these now by being part of the

61:11 So the F factor is not So it's still an F. Plus

61:14 . Okay. We call it HFR . Because it's integrated into the into

61:18 chromosome. Okay. And so uh it's still an F plus because it's

61:24 of transferring but um because now it's of the whole plasma. And so

61:30 it what happens is you have common . So I s it stands for

61:35 sequence. Okay. And the plasma chromosome have that in common. And

61:41 where these will integrate into the And so again remember the combination requires

61:48 level of hm Ology. And this what provides that. And so it

61:53 . And so now we have integrated . And now by being part of

61:59 chromosome down, the whole chromosome is . Now the whole chromosome won't be

62:06 . It's very rare that that ever if ever but you can copy chunks

62:12 the chromosome and that can be transferred another cell. Okay. So in

62:18 think of the chromosome now as a F factor plasma if you will.

62:25 . But the whole thing is copying begins to be transferred. Okay now

62:32 congregation of and HFR with an Plus cell. Okay, so when

62:40 begins to get replicated it goes in direction. Okay. What's in the

62:48 are the genes that make it Plus? The transfer genes, et

62:53 . Okay, this is the the part of that second. Okay so

63:04 sell recipient cell inherits this portion then would become an F. Plus.

63:13 . But again that rarely happens. for this reason. Okay, so

63:17 mentioned replication begins this way. And continues. Uh and this the

63:27 part the transfer genes, the F factor genes, that would be the

63:34 bit to go into the set in very last part that would go into

63:38 cell. So the cells have to together for nearly two hours for that

63:48 happen. Okay. And like I , that rarely if ever happens.

63:53 . So of course, what what recipient cell inherits is um certainly,

64:00 know, anything from from this point here to here to here to

64:05 All dependent on how long they're Okay, so certainly the F minus

64:11 benefits because it's inheriting, you potentially a bunch of different genes.

64:17 . Um it just doesn't have the to then transferred horizontally to another

64:22 Okay. But certainly it can pass . Right? It's gonna it would

64:28 to become re combined with the with host chromosome. But the main point

64:33 want to make here, is that why an F minus recipient won't become

64:37 F plus when it congregates what's in Because this is the last bit to

64:42 transferred and they'd have to be together long time and it just doesn't it's

64:46 not reality, there's different forces. when it sells are, you

64:50 in whatever environment they're in, they're bombarded by molecules that knocking

64:56 Remember brownie and movement. Brownie movement not really motion. But it's these

65:01 of vibrate Torrey motions that occur because are bouncing off of them or other

65:05 are bouncing off of them. Um are all forces that will break the

65:10 . Okay so to be together for two hours is not a likely

65:17 Um So here again we see again F minus made. Okay so so

65:26 so has the integrated uh F plus in here and then congregates with F

65:35 . So so we're looking at um has he's able to synthesize veiling veiling

65:42 synthesis pathway via L. Plus. what that means. The F minus

65:47 is incapable of doing that. So it's minus for that. And so

65:51 conjugate and um and you can see the the criminal is being replicated and

66:00 that copy is being pushed into the cell and if they're locked together long

66:06 maybe the railing gene goes into Or railing Operandi goes into it.

66:13 . An investigation that combines and now F minus cell which used to be

66:18 minus is now beginning plus. Okay so again it's not it doesn't become

66:25 plus but it's not necessarily a negative now it has acquired this ability to

66:30 bailing which which would be a Okay so um the uh of course

66:38 H. Bar still stays as Plus obviously. So um any questions

66:44 that? Yeah. Yeah correct. an F minus sell um will have

67:00 specific receptor that F Plus cells do . Okay so enough Plus cell that

67:06 will be specific for that receptor. it only will bind with an F

67:10 but that initial binding also prevents another plus cell from conjugating with it.

67:18 it's going to be very specific between those two things and like you said

67:22 direction. Yeah um Many of Okay so let's um look at

67:35 This is a this is about the . Prime factor from it. And

67:39 again um let me turn this thing here. So again remember that?

67:48 so recombination again plays a part in process. Okay um and let's see

67:57 we Can get this one. Okay Pepsi. Okay let's see what

68:58 get. Okay um That was magically like when that happens. All right

69:09 um so it is two and What are the two? What's

69:13 Yeah I think it's seen Chf are yes and yeah it involves excision.

69:23 it's about so plants can not only to formulate a far so they can

69:28 come out okay as well. What don't know is the frequency at which

69:36 occurs. I don't think it's it's I'm not exactly sure but but it's

69:43 the F prime is all about the part. Okay so here's an HFR

69:51 and so the main part to focus is the integrated plans with their the

69:57 part. Okay. Between the two sequences. Okay so again this stuff

70:03 here? right? That's that's the , right? Were inserted.

70:08 So um what happens is it can , exercise needs to come out of

70:17 chromosome and normally it comes out what out and what went in as you

70:22 there. So the box part is integrated in the first place. And

70:26 normally is what comes out. to form the the intact Klansmen

70:32 uh now that that's what I don't . The frequency of, I'm not

70:35 what the frequency of the HFR cell when it then comes out again,

70:38 don't know. But um but even is the event where when it comes

70:46 , I I used the term cockeyed the parent is what the textbook

70:52 right? So what that means is shifted. So here we're taking out

70:58 within the box, but we're taking this box instead. Okay, so

71:06 taking that. So we're leaving this part stays behind. Right? And

71:12 part with the B gene is now . A new part of the plasma

71:19 . Okay, so this is gonna our new plasma, What you see

71:26 . Okay, so once again, the chromosome or gene, right?

71:32 wasn't initially part of that. I when it wasn't serving. Okay.

71:37 so but now it is it's a that's what we call the F prime

71:41 contains. So if it weren't if just f we just say F

71:46 for example. Okay. it would be that would just be the purple

71:52 back into a circle. Alright. because it went the skew, so

71:58 speak, it's an F prime. it's carrying now basically it's carrying

72:04 It didn't have before when it went but now it does. Okay.

72:08 that's the F prime factor. So then I remember that part of

72:14 part of the plasma is left behind chromosome. They're so focused on the

72:20 . The parts of the purple are of the original plasma that integrated and

72:26 end result now is part of that is left behind. But then it's

72:32 part of the genes from the chromosome in this case. Okay, so

72:39 the big deal about that? um what can happen is so see

72:45 prime is self contained. The Prime plasma congregates with with another cell

72:51 population. Okay, it means that that new cell now may acquire an

73:01 copy of that gene. Okay, if they're members of the same species

73:07 they both already have the B Well now it requires a plasma containing

73:10 B gene. What that means is what we call a partial deployed.

73:16 So partial deployed is the type that have full we contain have genes from

73:25 have genes from her father. The deployed has only you know, two

73:30 more one or more genes are Okay, so that's a partial

73:34 So here we have to be copies gene copies part of the no no

73:40 deployed anywhere else. Just with that respectively. So what happened in this

73:45 is the B. Gene here can evolve independent, right? Because it

73:54 whatever the function of the B. here is then that's what it will

73:58 to do. Right? But now got this other one that doesn't have

74:02 same selective pressure on you can mutate a higher rate, right? Because

74:08 really not just it's just a secondary of a gene there. Right?

74:12 maybe it acquires a mutation or two it requires a slightly different function perhaps

74:18 it can be a benefit. Okay it's okay to get two copies of

74:22 gene where one can um evolving the . Okay um and so let's this

74:32 kind of puts it all together here terms of conjugation. So we had

74:38 our our basic f minus F plus . Okay so F F minus.

74:45 recipient receives a classic F f. becomes f positive f minus becomes

74:51 Right then we have the HFR So fact that integrates can I? And

74:58 meeting with f minus cell generally means the f minus stays epitomized because um

75:06 together long enough to transfer that last of information and that's where the F

75:10 is. Okay but it certainly can um some new uh one or more

75:17 along the way. Okay. Whatever are on this segment it's inheriting.

75:22 . So then finally the F Right? So they have prime formation

75:28 an HFR cell. Okay. But occurs due to this weird weird

75:34 Right? So in this example we a as part of our chromosome.

75:40 . Are non planted gene and then the apparent excision it's now on our

75:47 . But. Okay, so the variations here if you will.

75:52 so you see where you know the of course important here. Certainly integration

75:59 the F factor. That's recombination. we start a combination and transformation as

76:05 where that's important. So anyway, questions about that? Okay.

76:12 So for the mm. Mhm. that's minus cell. Well, the

76:28 , I can see the picture is little misleading. So the so this

76:31 here, Right. Uh it wouldn't initially have that plasma in it.

76:38 would be plasma less. Okay, it would only have the chromosome sitting

76:44 agent. Um So having following what's on. Alright, so the plasma

76:52 integrated in red. Right? It the genius to transfer. But now

76:59 it's part of the chromosome, the gets mobilized. Okay, so now

77:05 copying the chromosome and as the copy that is funneled into the recipient.

77:15 . And depending on how long we're determines how much the chromosome gets

77:22 The part that gets transferred. All . It was going to exist in

77:26 f minus cell. It's going to to recombine, Right? That's little

77:31 the extra combining with that. F minus. So that becomes part

77:35 that F minus chromosome ken. So instead of being a plasmid,

77:43 outside of chromosome, it's integrated into minus chromosome because that's where the

77:49 They have the age of our I have a plasma. They have the

77:53 plasma and then it transferred that in fashion. Um, so we've got

78:00 . So see you next. See monday. Okay. Mm hmm.

78:14 , I'm still a little I didn't to stop up class. Okay.

78:20 little just a little while. What's on here? So I know that

78:24 cf, there's enough of them and . It comes in here. It

78:29 competence factor action. Already explained

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