Bill comments:

BActeria have sex all the time.  They can deliver DNA to all one another all the time.  The question is, if you have bacteria that takes up DNA, it doesn't want to take up DNA from other bacteria because this could kill you.  You want to have an immune system that kills phage that comes from other organisms.  The way that bacteria prevent this is by using restriction modification systems. 

One of our favorite restriction sites comes from Ecoli, it is ECOR1.  it recognizes gale and abby throw their cat.  GAATTC.  So if this is the upper strand, what is the bottom strand? 

  GAGAATTCAA-3'

5'-CCCTTAAGTT

A restriction enzyme has eyes, where it can a sepcific sequence.  It has hsdS -specificity.  The protein has 2 eyes...the hsdS unit.  At another level, in a hand, it has a hammer.  We will call this a modification subunit.  In another hand, we have scissors.  These are both controlled by the eyes.  Imagine if you are a bacteria, and you have some phage shooting it's DNA.  You don't want to be infected with that.  Phages, for the most part, are bad.  They will kill you.  You need a way and if it scans that sequence, it knows to cut...

GAG                AATTCAA

CCCTTAA                GTT

These are phosphate bonds between each sequence area, and the sticky ends can be seen.  In fact, the cohesive ends all see different sequences.  here is the problem.  If you recognize a sequence, why don't you just cleave your own DNA?  CHiara: you wouldn't exist any more.  Bill: You'd be dead!  You'd fall apart.  How do you prevent the cleavage of your own DNA?  Genevieve: another enzyme?  Bill:  the hammer.  It goes ahead and hammers on a methyl group onto it's own DNA.  If it sees the methyl group, it says that I am not going to cleave it.  So, the eyes allow you to see a piece of DNA...and it can see a specific DNA sequence.  When it replicates it's own DNA, it puts methyl groups on.  If it doesn't see the methyl group, boom, you are clean.  Isn't that amazing...it's like a built in immune system to protect itself from phages.  Bernard Arter? Got his noble prize for discovering this.  He did a simple experiment that you can understand...you know I never go thru this...it's amazing that all these things we need to know...all of these guys won noble prizes, theyare fundamental biology concepts.  He was studying probably the most famous phage of e.coli.  K-12 E.coli, everybody used this for bacteriogenetics, it happens to have a natrual phage in it, called bacteriophage lambda.  Greek lambda.  lambda(k) would plaque...picture..grid:

                    k            B            C

lambda (K)    10^10  10^6      10^10                      # of plaques as determined by high titer.

lambda (B)    10^6    10^10    10^10

lambda (C)    10^6    10^6      10^10

he ended up discovering that K12 has a different restriction enzyme system than B.  They have different sets of eyes.  C doesn't have one.  It can't decorate it's DNA.  It can't see anything as foreign, it has no restriction system.  Plaquing another phage on it and it plaques just fine.  C has no restriction system.  You can imagine then that all the different restriction enzymes just recognize different sequences. 

Cla...  First letter is the genus, La is two letters for the species, ... does all this make sense for how this works?  ...SMith? got noble prize for understanding how the sequence is.  Let's say these sticky ends are from E.coli.  Do you see that if you cleave celery with EcoR1 that you can get the same sticky end?  Recombinant DNA, just mixing DNA with DNA from celery, and you can get recombining DNA.  You can recombine DNA from any organism.  This is what allows us to do recombinant DNA.  Most DNA's, i.e. rat gut, and celery, and they will have compatible ends.  Or, put luciferase into a phage. 

Bill says that the restriction system is dependent on the host system that we are growing in...it is the smegmatis that will have it's own restriction system.  We are not using 3 different E.coli.  Every phage will have a different

Bill recommended that we use the program clone, cut bacteriophages...use the lab copy that the group liscense where we can use it, learn how to clone and restriction ...we can predict the bands that will arise from our anticipated cutting, later. 

Genevieve: how to calculate # of cut sites?  Bill: to determine that, any chance of having a base pair is 1 in 4.  1/4 x 1/4 x 1/4 x 1/4 x 1/4 x 1/4 x 1/4 = 1/4096.  This is the probability that this sequence shows up.  So if your genome is 50,000 base pairs, how many times should it cut?  In fact, it changes if the GC content is only 50%.  It only happens if 50%.  65% G-C mycobacteria, other bacteria are 50%.  How would this effect your outcome?  See if you can determine this. 

Biss suggested that we use BSTEII and