Bacterial Plasmid Transfer

Plasmid Sex

Introduction

.....In most bacteria there are several pieces of DNA. .. One is the somatic genome - a huge circle of double-stranded DNA that actually measures about 2 mm in length, and is all crammed into the little cell. ..This large piece of DNA is what defines the type of bacterium it is. .. The cell cannot live without this circle of DNA. In addition, there are various optional smaller circles of DNA, which are usually called plasmids... To repeat: ..these are 'optional' and the cell can get along without them unless the genes on those plasmids allow it to survive under unusual conditions such as when a particular antibiotic is in the neighborhood, and the plasmid contains a gene that protects the cell from that antibiotic by any one of several different mechanisms.

.....A cell can duplicate a plasmid and then send one copy over to another cell via a thin tube called a pilus. ..Seemingly, each type of plasmid codes for its own special type of pilus.

.....A widely used plasmid of E. coli is one called "F" (for fertility). .. Cells that possess "F" are called male (F-donors or "F+"), these cells usually possess two F-pili for the transport of the F into cells lacking it. .. Those without F are called females (potential recipients, F-). .. In this exercise you will mix a very few F+ with a much larger number of F-, and show that the F- cells are converted to F+ at a rather rapid pace. ..The rapidity is because because it is a chain-reaction as the newly form F+ cells are added to the pool of donors. ..(You can change this exercise into an experiment if you can design an experiment from which you can determine the time it takes to convert an F- into an F+.)

.....First, you must have a strain of donor that possesses a somatic genome that is different from that of the recipient. ..That way you can "select against" the original donor cells by using a mating medium in which they cannot grow, but in which the recipients can. ..For this we will use donors that are normal with respect to sensitivity to the antibiotic streptomycin, while the recipients will be streptomycin resistant (strR). ..We will add streptomycin to the medium, and none of the original donors will be able to grow up to form colonies. ..(Always keep in the back of your mind a very important word: "controls"!)

.....Second, you must have a way to tell whether or not the recipients have acquired the F. ..We will do a little trick here, and use an F that has the lactose operon inserted into it. ..This is called "F-lac." ..(Whenever other genes are incorporated into F, the F is now called a type of F-prime (F'). ..Thus F-lac is a type of F'. ..We will therefore start with F- cells that do not have a functional lac-operon (lac-), and so when they acquire the F-lac, they will be able to use lactose sugar to grow. .. (This is not a genetically engineered product, but one that can be made naturally, and therefore doesn't not need to be approved by an institutional recombinant DNA committee. ..Indeed, no part of this exercise is considered genetic engineering. ..It is all genetic recombination using natural means. ..That is one of the important things about this exercise - this happens naturally around you all the time in the genetically mobile and fluid microbial world.)

.....Back in the first decade of the 1900's, Dr. MacConkey concocted a medium bearing his name. ..On MacConkey agar (page 61), E. coli that possess the ability to ferment lactose (lac+) grow up as red colonies, while lac- mutants grow up as pale colonies. ..Thus what do we need? ..Yes! ..MacConkey agar into which we have added some streptomycin. (Controls?)

THE EXERCISE

  1. Make two separate overnight cultures of the donors and recipients in 0.7% tryptone (a la page 2). .. (Please don't follow the directions on the bottles! ..Use TAP-water as these little critters need minerals to grow.) .. Do NOT add any sugars to the medium!
  2. Into a new sterile bottle of 20 ml of tryptone add 20 drops of the recipient F-, and 1 drop of the donor F-lac. ..(Here strict aseptic technique is not required because the product of this reaction will be plated on antibiotic plates.)
  3. At timed intervals make plate counts (page 112) of the culture using the MacConkey agar PLUS streptomycin.* ..(The next day, your results should show many pale colonies on the plates, but as time proceeds there will be more and more red ones, which are clones of the recombinants.)

    * Making antibiotic medium is a little tricky as most antibiotics are destroyed in heat, and therefore cannot be autoclaved. ..Of course you are wondering why worry about sterilizing an antibiotic in the first place. .. Because they are not free of bacteria! ..They are either made by a type of bacteria or fungi, which will then contaminate the powder. ..Thus the antibiotic powder must be dissolved in water at the desired concentration, and then sterilized. ..The most common way to do so is by "ultra-filtration" through sterile filters that have such small holes that bacteria cannot get through while the antibiotic molecules easily fit. .. The filtrate is collected in a cool pre-sterilized container. ..The most common devices are little filters that fit onto the ends of large hypodermic syringes. .. The syringe (without the filter) is filled with the antibiotic solution, and then the sterile filter device is screwed onto the bottom of the syringe... The liquid is forced through the filter by pushing down on the syringe piston. ..The stream or drops of sterilized antibiotic solution is then allowed to flow into a sterilized bottle that can the be screw capped tight. .. A popular brand of such filter assemblies are 0.2nm AcroDisks®, and are sold through most laboratory supply houses. .. (Virus suspensions and heat labile vitamins are also sterilized in this same way.)

RETURN TO TOP OF PAGE

I WANT TO ESCAPE THIS PAGE!