BCOR11 Laboratory 7: Bacterial Transformation I Plasmid Isolation & DN !

!is"ali#ation O"tline of cti$ities and Ob%ecti$es I& Bacterial Plasmid Isolation 'Re$ie( im)ortant safety )reca"tions '*nderstand (+at a bacterial )lasmid is and (+at it means to transform bacteria ',ain )ractice isolatin- bacterial )lasmids !is"ali#ation of .o"r Plasmid: -arose ,el /lectro)+oresis '*nderstand t+e basic )rinci)les of a-arose -el electro)+oresis 'Learn +o( to )re)are an a-arose -el ',ain )ractice $is"ali#in- DN sam)les 0i&e& )lasmids1 "sin- a-arose -el electro)+oresis Disc"ssion of Protein 2am)le 2e)aration by P ,/ 0contin"ation of Lab 31 'Com)are and contrast )roteins isolated from t+ree different so"rces 'Demonstrate yo"r "nderstandin- of )olyacrylamide -el electro)+oresis

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In todays lab, you will begin a series of experiments designed to introduce you to a fundamental laboratory technique: bacterial transformation. Bacterial transformation is a process by which circular pieces of DN called plasmids are !inserted" into bacteria. #hese circular bits of DN are copied by a bacteria e$ery time it di$ides along with the bacterial genome. s you %now, genes contain instructions for the production of certain molecules, most often proteins. ny genes contained in the plasmid are expressed in the bacteria, which means that the bacteria follow the instructions in the genes and produce the specific proteins. &sing bacterial transformation it is therefore possible to introduce new genes into bacteria that allow them to produce new proteins. #hese proteins often gi$e them additional characteristics, for example the ability to grow in the presence of an antibiotic. #his wee% '(ab )*, you will isolate and then $isuali+e a bacterial plasmid from one strain of E. coli that a contains a gene which confers resistance to an antibiotic, ampicillin. In (ab ,, you will transform this plasmid DN into a different strain of E. coli that does not contain a plasmid and is not resistant to ampicillin, -ou will plate these transformed bacteria on plates with and without ampicillin, including proper controls to assess whether your transformation was successful. t the beginning of (ab ., you will be able to loo% at your plates and determine, comparing your transformants and controls, whether your transformation wor%ed.. /ead the introduction to !Bacterial 0lasmid Isolation" below for further details about bacterial plasmids and the general principles of bacterial transformation.

Bacterial Plasmid Isolation I102/# N# 3 45#- N2#53 #he recombinant 'human6constructed* DN used in this experiment requires a 07 le$el of containment. #his means that you must obser$e the following safety precautions: 8ear glo$es at (( times. bsolutely N2 eating or drin%ing in the lab 'as usual*. 8ash your hands B542/5 you lea$e the lab room, e$en if you 9ust step out for a moment. N- materials that touch the bacterial culture 'pipettes, glo$es, etc.* must be placed in the red bioha+ard bags, which will be autocla$ed before disposal. #his includes plates, toothpic%s, and microfuge tubes. Do not put materials such as paper towels or regular garbage in the red bioha+ard bags. &se pipetting de$ices and micropipettes to dispense all liquids. bsolutely no mouth pipetting allowed.

Introduction s described in the 2utline of cti$ities and 2b9ecti$es, plasmids are circular pieces of DN that can be used to introduce new genes into bacteria which allow them to produce new proteins that you, the researcher, want them to ma%e. 4or example, researchers might transform bacteria with a plasmid that contains the human insulin gene, along with a gene for drug resistance, in order to collect large amounts of the insulin that the bacteria produce as they grow and multiply. lmost always, plasmids that are transformed into bacteria also contain genes that confer resistance to specific drugs such as ampicillin. Including antibiotic resistance genes in plasmids allows researchers to determine which bacteria ha$e been transformed with the plasmid, and which bacteria lac% the plasmid by simply growing the bacteria in the presence of the antibiotic. Bacteria that contain the plasmid will be able to grow in the presence of the antibiotic 'ampicillin*, while those that do not contain the plasmid will die. #his process is called selection. -ou !select" the bacteria with the plasmid by growing them in the presence of the antibiotic. In this lab, you will isolate a plasmid that contains an ampicillin resistance gene and , the next wee%, transform it into a non6resistant strain of E. coli 'see figure below*. #he gene that confers resistance to ampicillin, which is $ery commonly used for selection, allows the bacteria to express 'produce* an en+yme that degrades the antibiotic. #his pre$ents the ampicillin from brea%ing down the bacterial membranes of any bacteria that express this gene.

0lasmid Isolation #he day before your lab, the lab coordinator will start growing cultures of E. coli that contain a plasmid called pB/:;; that contains the ampicillin resistance 'see the ppendix for a description and diagram of the plasmid*. #he bacteria will be grown o$ernight in (B broth 'basically bacteria food* with ampicillin in an incubator where the temperature is %ept at :)<= and the tubes containing the cultures are continuously sha%en. '3ha%ing promotes growth of the bacteria by mixing up the culture to distribute the bacteria more e$enly in the food source and aiding oxygenation.* &sing the culture pro$ided by the lab coordinator, perform the steps below to isolate the plasmid containing the ampicillin resistance gene. 7* #ransfer 7.> ml of the culture to each of two microfuge tubes ;* =entrifuge the two tubes for fi$e minutes in a microfuge :* =arefully pour the supernatant 'liquid* into a bea%er containing a 7?@ bleach solution A* dd 7?? B( of 3olution I to each tube

>* &se the broad end of a flat, sterile toothpic% to gently resuspend the pellets C* Incubate at room temperature for fi$e minutes )* dd ;?? Bl of 3olution II to each tube and mix by in$erting the tubes

,* Incubate on ice for > minutes .* dd 7>? Bl of pre6cooled 3olution III to each tube and mix gently by in$erting the tubes

7?* Incubate on ice for fi$e minutes 77* =entrifuge for > minutes 7;* Being $ery careful not to disturb the pellet, carefully transfer 2N(- the supernatant to a new tube 7:* #o precipitate your DN 'get it out of solution*, add 7 ml of cold .>@ ethanol to the supernatant, and place your tubes on dry ice for 7> minutes. DDDry ice is $ery coldE do not touch it with bare s%inDD 7A* =entrifuge your tubes for ;? minutes
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7>* =arefully remo$e the .>@ ethanol using a pipette F 3 G5 #H5 05((5# 7C* Iently wash the pellet by adding ;>? Bl of )?@ ethanol, centrifuging for fi$e minutes, and pouring off the ethanol 7)* ir dry the pellet by tapping the ethanol gently out of the tube and letting the tube dry for ;? minutes with the cap open 7,* Dissol$e the pellet in ;> Bl of #5 buffer and place tube on ice -ou will be using part of this sample today to run on a gel to $isuali+e plasmid DN 'see below*. #he other portion will be fro+en away and used to transform the non6resistant bacteria next wee% '(ab ,*. Save this portion of sample in the freezer!

!is"ali#ation of .o"r Plasmid: -arose ,el /lectro)+oresis garose gel electrophoresis is similar to polyacrylamide gel electrophoresis in that it allows you to separate molecules, in this case DN , based on their si+es. (i%e polyacrylamide, agarose forms a matrix that DN molecules mo$e through based on their si+e. ll DN is negati$ely charged, so when an electric current is applied, the DN molecules in the samples you load on the gel mo$e from the negati$ely charged cathode to the positi$ely charged anode. (arger DN molecules ha$e a more difficult time getting through the agarose matrix than smaller molecules, and therefore mo$e more slowly through the gel than smaller molecules. #o $isuali+e your DN , a molecule called ethidium bromide is included in the gel. 5thidium bromide binds to DN molecules and will fluoresce 'glow* when exposed to ultra$iolet light. I102/# N#: 5thidium bromide can bind to any DNA molecule including yours and therefore has the potential to cause changes to your DN 'mutations*. Handle the gels carefullyJ #he # or the lab coordinator will be preparing the gel. Iel and 3ample 0reparation 7* In a glass bea%er, mix ?.:> grams of agarose in >? ml of electrophoresis buffer. #his will gi$e you a gel that is ?.)@ agarose. '#ypical DN gels range from ?.>@ to :@ depending on the si+es of DN being separated.* ;* &sing K;? second !bursts," heat the agarose mixture in the microwa$e and gently swish until all the agarose has dissol$ed. 8atch closely and do not allow your solution to boil o$er. Handle the bea%er carefully using the protection pro$ided F its hotJ :* A* llow agarose to cool slightly K7? minutes dd ;.> Ll of ethidium bromide to the agarose mixture& C *TION: Remember to +andle t+e et+idi"m bromide caref"lly beca"se it can ca"se m"tations in yo"r DN 4 T+e T or lab coordinator (ill be )re)arin- t+e -el&

>* 0our the solution into the gel casting tray and place the comb into the top of your gel
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llow your gel to cool until opaque K;? minutes

)* 8hile your gel is cooling, prepare your DN sample by mixing A Bl of CM sample buffer with 7? Bl of your plasmid DN . Remember to save the remainder in the freezer for next ee!! Note: #he sample buffer contains sucrose to ma%e the sample dense, which causes it to fall to the bottom of the well when you load it, and a dye called Bromphenol Blue to gi$e the sample color that will allow you monitor the sample as it mo$es through the gel. ,* 2btain a tube of "ind### molecular weight mar%ers, which are already mixed with sample buffer

garose Iel 5lectrophoresis 7* 0lace the gel in the gel running box, carefully remo$e the comb, and co$er it with electrophoresis buffer ;* (oad your plasmid DN sample and the "ind### mar%ers on to the gel :* =onnect the electrodes so that your DN will mo$e from the wells at the top of the gel towards the bottom. 'Hint: /e$iew the introduction to this section if you cannot remember which direction DN mo$es and why.* Be sure that you ha$e the electrodes in the proper orientation or your DN sample will mo$e up through the top of your gel and be lost in the bufferJ A* /un the gel at 7?? G for A> minutes or until the blue dye is about halfway down the gel. >* #urn off the power pac%, carefully remo$e the gel, and place the gel in a Niploc bag. /emember that your gel contains ethidium bromide. Be sure that you are wearing glo$es before you touch the gelJ C* 0lace the lefto$er buffer in a plastic 9ug to be disposed of properly 'it now contains some of the ethidium bromide that was in your gel*. )* 8earing goggles to protect your eyes from the &G light, examine your gel using a &G light box. -ou should %eep your gel in the Niploc bag when $iewing it on the light box. ,* -our laboratory # will show you how to ta%e a photograph of your gel on the &G light box I4 #I15 D253 N2# 05/1I#, -2&/ # 2/ #H5 ( B =22/DIN #2/ 8I(( /512G5 #H5 I5( ND # O5 0I=#&/5.

Disc"ssion of Protein 2am)le 2e)aration by P ,/ 0contin"ation of Lab 31 In (ab C you separated protein samples isolated from three different sources 'fish, egg white, and li$er* using polyacrylamide gel electrophoresis '0 I5*, and you should ha$e recei$ed a picture of your gel from your
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laboratory # $ia e6mail. #oday you will wrap up the experiment with a brief discussion focusing on the following questions: 7* How did your gel runP 8hat si+es of proteins are found at the top of the gelP t the bottomP ;* 4rom your %nowledge of the molecular weight mar%ers, can you tentati$ely identify any of the protein bands in the samplesP :* Do the protein samples appear to be related when you obser$e their electrophoretic patternsP A* 8hat are the limitations of =oomassie staining as a technique to $isuali+e proteinsP In addition, to demonstrate your %nowledge of the basic principles of 0 I5, you will discuss the questions you answered for your (ab C post6lab assignment. Be sure to as% your laboratory # if you are confused about any part of the technique. In the (ab exercises you complete today, you will gain experience using a different %ind of gel electrophoresis. It is important that you be able to recogni+e the similarities and differences between the techniques. ))endi5 0lasmid Isolation 3olution 7 >? m1 glucose ;> m1 #ris6H=I, pH ,.? 7? m1 5D# ; mgQml lyso+yme 0lasmid Isolation 3olution ; ?.; N Na2H 7@ 3D3 0lasmid Isolation 3olution : C? ml >1 O2 c 77.> ml glacial acetic acid ;,.> ml dH;2 #5 buffer 7? m1 #ris6=l pH ).> 7 m1 5D# , pH ,.? CM DN 3ample buffer ?.;>@ bromophenol blue A?@ weight by $olume sucrose >?M # 5 5lectrophoresis Buffer ;A.; g #ris base >.) ml glacial acetic acid :.) g Na;5D# 6;H;2 pH to K,.>
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pB/:;; 0lasmid pB/:;; was created in 7..) and was among the first bacterial cloning plasmids to be widely used. It contains the gene that confers resistance to ampicillin as well as a gene that confers resistance to tetracycline 'another antibacterial drug*. pB/:;; has many specific DN sequences called restriction sites, some of which are shown in blue in the diagram below. /estriction sites are short sequences of DN that can be recogni+ed by specific en+ymes 'called restriction en+ymes* that cut the DN at a certain place within the restriction site. 1any of the restriction sites in pB/:;; are located in the ampicillin and tetracycline genes. /esearchers can use a restriction en+yme to cut the plasmid within one of the drug resistance genes. gene of interest 'li%e the human insulin gene* can be inserted into the cut site, and the plasmid !sealed" bac% up to form a circle again. Insertion of DN into a restriction site in either of the drug resistance genes causes the gene to become non6 functional. 0lasmids that contain the new gene can be isolated by transforming the plasmids into bacteria and loo%ing for bacteria that can grow in the presence of one drug, but not the other 'i.e. if the gene of interest is inserted into the tetracycline resistance gene, the bacteria would die in the presence of tetracycline, but sur$i$e treatment with ampicillin*. -ou will learn more about restriction en+ymes and their uses in (abs 7? and 77.

BCOR11 Lab 6 Pre'Lab ssi-nment D"e at t+e be-innin- of Lab 6 4or the experiments you perform in (abs ) and ,, you will be writing 'in pieces* almost a complete laboratory report: Introduction, 1aterials R 1ethods, /esults, Discussion, and /eferences. In (ab , you will wrap up your !tour" of the sections by discussing Discussions. #o prepare for this exercise, complete the reading assignment below and answer the questions completely, but succinctly. In addition, you should write short outlines of the Introduction and 1aterials R 1ethods sections 'see below*. 0lease type or neatly write your answers on a separate sheet and number the questions appropriately. -ou can include your outlines with the questions. $e sure to put your name% lab section% and &A on your assignment. #his assignment 'questions and outlines* must be completed by the beginning of the next lab period '(ab )*.

/eading ssignment: 6Onisely, O ';??.* student handboo% for writing in biology :th edition" Discussion 'pages );6)>* 6/e$iew pages 7;:67:> for good tips on writing and re$ising

Suestions 7* 8hat is the main difference between the /esults and Discussion sections of a laboratory reportP ;* (ist three questions you should address in a Discussion section. :* &nexpected results are typically addressed in the Discussion section. Imagine that you did not see a band on your agarose gel when you tried to $isuali+e your plasmid 'or perhaps you actually didnt*. How might you explain this resultP

Bacterial Introduction and 1aterials R 1ethods 2utlines lthough you ha$e not yet completed the second part of the Bacterial #ransformation experiment where you will transform your isolated plasmid into a different strain of E. coli and grow up the cultures in the presence of ampicillin to determine whether your transformation was successful , you should start thin%ing about writing the Introduction and 1aterials R 1ethods sections, which are due at the beginning of (ab .. In preparation for writing these sections, you should write brief outlines of each section. #he goal of these outlines is to help you organi+e your thoughts, not to lay out all the details you will include. 4or the Introduction, thin% about what %ey pieces of bac%ground information you will need to include, what question you were trying to answer with your experiment, and why you performed these experiments a particular way F it is not acceptable to state that it was a required exercise for the courseJ*. 4or the 1aterials and 1ethods section, 9ust list the $arious techniques you used, which can ser$e as headings to organi+e the section 'i.e. growth of bacterial culture*. -our outlines should be included with your answers to the questions abo$e.