[Universidade de Evora]

UNIVERSIDADE DE ÉVORA

DEPARTAMENTO DE BIOLOGIA

LABORATÓRIO DE BIOLOGIA MOLECULAR

Responsável: Prof. Carlos Sinogas

UNESUL - Parque Industrial de Évora
Rua da Barbarrala, 1 - 7000 ÉVORA

Tel./Fax: 266 750 617 / 8
Email:
labbiom@uevora.pt

Engenharia Genética

Clonagem de DNAs em Bactérias

(Módulo I)

Acção Foco 1999 - Programa Prático

 

Carlos Sinogas, Luís Alho, Isabel Brito, 1999

SUMÁRIO

OBJECTIVO *

CULTURA DE BACTÉRIAS em meio líquido *

Preparação de meios Sólidos *

Extracção e Purificação de DNAs (MiniPrep) *

PREPARAÇÃO DE DNA PLASMÍDICO - (Qiagen) *

quantificação de DNAs *

RESTRIÇÃO Enzimática *

Separação Electroforética *

"sabe clonar um Dinossáurio?" (Trabalho adaptado de Kit educacional da Stratagene) *

"Transformação Bacteriana" (Trabalho adaptado de Kit educacional da Stratagene) *

  1. OBJECTIVO

 

 

 

Material Biológico

Bactérias transformadas com plasmídio vector (pSK+) e com plasmídios recombinantes

sumário

CULTURA DE BACTÉRIAS em meio líquido

Reagentes

LB 10X:

Bacto-triptona 100 g

Extracto de levedura 50 g

NaCl 100 g

H2O destilada até 1000 ml

pH 7,5. Autoclavar. Conservar estéril a 4C.

Ampicilina 1000X:

50 mg/ml em água. Esterilizar por filtração, conservar a -20C.

Água destilada e esterilizada

Protocolo Experimental

- Preparar assepticamente 50 ml de meio LB 1X com água estéril
(45 ml de água + 5 ml de LB 10X).

- Adicionar 50 m l de ampicilina 1000X (50 m g/ml).

- Inocular com bactéria contendo o plasmídio a preparar.

- Incubar 37C durante uma noite sob agitação.

sumário

Preparação de meios Sólidos

 

Reagentes

LB 10X

Ampicilina 1000X

Água destilada e esterilizada

Agar agar (Difco 80-120 Mesh)

 

Protocolo Experimental

Agar 2X

- Pesar 3,0 g de agar para fraco rolhado autoclavável de 250 ml

- Adicionar 100 ml de H2O destilada

- Aquecer a 100C até completa dissolução do Agar

- Autoclavar a 121C durante 15 minutos

- Deixar arrefecer e fechar bem os frascos. Conservar estéril.

Agar - LB/amp

- Preparar assepticamente 100 ml de LB 2X (20 ml LB 10X + 80 ml H2O)

- Adicionar ampicilina 1000X (200 l) se necessário

- Aquecer a cerca de 50C em banho-maria

- Fundir Agar 2X em forno microondas
(não usar potência máxima e afrouxar ligeiramente a rolha do frasco)

- Adicionar ao frasco com Agar 2X fundido (100 ml), os 100 ml de LB 2X

- Homogeneizar evitando a formação de bolhas de ar

- Colocar em banho-maria a 45C até utilização

Placas de Petri

- Sem abrir totalmente a placa, verter cerca de 20 ml de meio a uma temperatura de 45C, tapando a placa de imediato

- Após a solidificação inverter as placas

- Conservar em frigorífico

sumário

Extracção e Purificação de DNAs (MiniPrep)

Reagentes

LB 1X com ampicilina.

Lisozima (pó)

Isopropanol

5 M NaCl

Etanol puro e 70 %

TE pH 8,0:
10 mM Tris-HCl pH 8,0
1 mM EDTA

Pronase 20 mg/ml em água (auto-digerida durante 2 horas a 37C. Conservar a -20C)

Pronase Mix:
Pronase 50 l
10 % SDS 86 l
H2O 860 l

RNAse A 10 mg/ml em:
10 mM Tris-HCl pH 7,5
15 mM NaCl
(incubada por 15 minutos a 100C e arrefecimento lento. Conservar a -20C)

RNAse mix:
RNAse A 40 l
TE pH 8,0 10 ml

Solução I:
25 mM Tris-HCl pH 8,0
50 mM Glucose
10 mM EDTA

Solução II:
0,2 N NaOH
1 % SDS

Solução III:
5 M KOAc 60 ml
Ac.acético glacial 11,5 ml
H2O destilada 28,5 ml
pH 4,8

Fenol:clorofórmio:
Fenol destilado 50 ml
Clorofórmio 50 ml
Álcool isoamílico 1 ml
8-hidroxiquinoleína 0,1 g
TE pH 8,0 15 ml
(usar apenas a fase orgânica, de cor amarela, não agitar)

 

Protocolo Experimental

- Inocular 5 ml de LB com cada colónia a analisar.

- Incubar a 37C durante uma noite com agitação.

- Pipetar 1,5 a 2 ml de suspensão para microtubo.

- Centrifugar 1 minuto na microcentrífuga.

- Decantar e escorrer completamente o sobrenadante.

- Ressuspender o sedimento com 100 m l de Solução I. Misturar.

- Adicionar 200 m l de Solução II. Misturar por inversão. Agitar.

- Repousar 10 minutos no gelo.

- Adicionar 150 m l de Solução III. Misturar.

- Agitar fortemente à mão para partir DNA cromossomal.

- Centrifugar 5 minutos na microcentrífuga.

- Pipetar 400 m l de sobrenadante límpido.

- Adicionar 250 m l de isopropanol. Repousar 10 minutos à temperatura ambiente.

- Centrifugar 20 minutos na microcentrífuga.

- Decantar

- Lavar o precipitado com etanol puro.

- Centrifugar 5 minutos na microcentrífuga.

- Decantar, escorrer e secar.

- Dissolver com 50 m l de RNAse-mix.

- Incubar a 37C durante 30 minutos.

- Usar 10 m l para digestão com enzima de restrição.

(50 g/ml = 1 UDO)

sumário

Quantificação de DNAs

 

Reagentes

Solução padrão de DNA (0,5 mg /ml)

Solução de DNA a quantificar

Parafilm

TE/BrEt: TE pH 8.0 contendo Brometo de etídio a 0,5 g/ml

 

Protocolo Experimental

- Preparar 6 diluições decimais do DNA padrão:

Tubo

TE/BrEt

DNA

Concentração

Amostra

Volume

1

45 l

Padrão

5 l

50 g / ml

2

45 l

Tubo 1

5 l

5 g / ml

3

45 l

Tubo 2

5 l

500 ng / ml

4

45 l

Tubo 3

5 l

50 ng / ml

5

45 l

Tubo 4

5 l

5 ng / ml

6

50 l

-

-

0

 

- Diluir amostra de DNA a quantificar a 1:10 com TE/BrEt

- Distribuir I gota de cada diluição do padrão, em fila, sobre parafilm

- Aplicar I gota idêntica da diluição do DNA a quantificar

- Observar sob luz UV

- Comparar as intensidades de fluorescência relativas

sumário

RESTRIÇÃO Enzimática

Reagentes

Solução de DNA a analisar

Enzimas de restrição:

Bam HI (10 U / l)

Eco RI (20 U / l)

Sal I (20 U / l)

Fenol:clorofórmio

Tampão Universal (Stratagene) 10X (TUS 10X):

1M KOAc

250 mM Tris-Acetato pH 7,6

100 mM MgOAc

5 mM b -Mercaptoetanol

100 m g/ml BSA

Protocolo Experimental

- Em microtubo, adicionar sequencialmente:

- H2O estéril 19,25 l (ou q.b.p. 25 l final)

- Solução de DNA 1 l (1 g)

- TUS 10X 3,75 l (1,5 X final)

- Solução de enzima 1 l (10 Unidades)

- Misturar no vortex.

- Centrifugar brevemente para levar tudo para o fundo do tubo.

- Incubar a 37C durante 1 hora.

- Desproteinizar pelo fenol:clorofórmio:

- Adicionar 25 l de fenol:clorofórmio. Misturar bem.

- Centrifugar 1 minuto na microcentrífuga.

- Pipetar fase aquosa para novo tubo.

- Tomar 10 l para análise em gel.

sumário

Separação Electroforética

 

Reagentes

TBE 10X

890 mM Tris

890 mM Ácido bórico

20 mM EDTA

pH 8,0

Agarose média ou baixa EEO (Sigma Tipo II ou V)

Brometo de etídio 10 mg/ml

Dye-DNAs (Indicador de Migração):

25 % Ficoll 400

0,25 % Orange G

DNA marcador:

lambda X Bst EII

 

Protocolo Experimental

- Fundir a agarose (0,7%) em 95 % volume final de água.

- Arrefecer a cerca de 50C.

- Adicionar 5 % de TBE 10X (0,5 X final).

- Adicionar 0,005 % Brometo de etídio (0,5 g/ml final).

- Misturar e verter no molde.

- Colocar o pente e deixar solidificar.

- Colocar o gel no aparelho, submerso em TBE 0,5X.

- Aplicar as amostras de DNA, contendo 10 a 20% de Dye-DNAs.

- Aplicar uma amostra de DNA marcador (0,5 a 1 g)

- Proceder à separação electroforética (75 V), até conveniente migração.

- Visualizar sob luz UV (l = 300 a 320 nm).

- Fotografar o gel com filtro vermelho de gelatina.

sumário

"Sabe clonar um Dinossáurio?" (Trabalho adaptado de Kit educacional da Stratagene)

Student Information and Instructions

Primary Objective

To perform a restriction digest and then ligate the resulting DNA fragment into a vector; conclude by confirming the ligation by gel electrophoresis.

Other Objectives

1. Define: DNA, restriction enzyme, cloning, ligation, insert, vector

2. Compare the functions of a restriction enzyme to DNA ligase

3. Review the Structure of DNA

4. Review the function of restriction enzymes and ligases in cellular function

5. Explain the importance of ligase in DNA replication

Scenario

A harsh light bathed the surface of the workbench with illumination. A door opened and a technician moved to the lab bench. She placed three frosty vials labeled, "Dino C-200-345e," "Amph R-100-000a," and "T4 Ligase," in an ice bucket on the bench surface.

The technician, Cynda Wilcox, took off the padded gloves she had been wearing to protect her hands from the super-cold containers she had brought from their storage in the -80C freezer. She flipped a switch over her work area. Immediately a rotating yellow light flashed in the hallway of the Wundergene, Inc. Biotechnology complex. Just below the light, a red neon sign glared its message, "Sterile Field - DO NOT ENTER!" The magnetic lock on the door beside the sign clicked.

After putting on latex gloves, Cynda opened the tube containing dino DNA. Using a micropipettor, she placed a small amount of DNA to a new microfuge tube and then placed the DNA on ice. She repeated this until she had 10 tubes with the same amount of dino DNA. She opened a second container and pipetted a set amount of the amphibian DNA from that container into the ten microfuge tubes. After adding a buffer to each tube, Cynda added an enzyme, T4 Ligase, and mixed the contents of the 10 tubes.

Cynda placed the tubes in a microcentrifuge and spun them for several seconds so the liquid went to the bottom of the tubes. Then she pushed the microfuge tubes through the holes in a foam floatation device and placed the tubes into a water bath and set the timer for a 60 minute incubation.

* * * * *

Two months later, in a different laboratory of the Wundergene, Inc., another technician watched closely as the egg tooth of a baby dinosaur pushed its way through the synthetic shell. When the entire body of the small creature lay panting beside the remnants of the shell, the lab technician scooped up the animal and placed it into the moist environment of a 40 incubator.

The little dinosaur perked up quickly in its new home and was soon hopping around, exploring its environment. The technician reached into the incubator and clipped the identifying band around the left rear leg of his charge. A snap of the needle-sharp teeth of the dinosaur just missed the finger of the technician. He hastily closed the incubator hatch. This Compsognathus was the most active yet. He made a notation in his lab book and placed a call to Cynda Wilcox.

"Cynda, this is Jesse, over in the hatch lab."

"Yes, Jesse, what do you need?"

"Nothing. Just have some information for you. Whatever you stuck into that dino DNA in batch, uh...," he paused to check the notation in his lab book, "Um, batch C-200-345e really caused a big change in the behavior of the critters."

"What kind of change?"

"The Compsognathus that just hatched is a lot more active than the others."

"That's not bad is it?"

"No, except this little sucker tried to bite my finger off. You sure you glued the right genes into those missing spots?"

"As sure as we can be. I'll check the notebook and see if there is anything extra special about the amphibian DNA we used to patch the holes."

Cynda turned back to her lab bench. Jesse turned back to his newest miracle, a young carnivore, whose closest ancestor had been extinct for over 65 million years.

Introduction and Background Information

The technology necessary to "glue" genes from one species into DNA from another has been around since the mid-1970s. While it is possible to create hybrid DNA and to amplify (make multiple copies) that DNA sequence millions of times, cloning an extinct creature from pre-historic DNA is not possible or even probable in the foreseeable future. There are just too many difficulties to overcome and variables to be controlled.

One, dinosaur DNA only exists as incomplete fragments. Entire dinosaur chromosomes have broken apart in the millions of years since the last dinosaur died. Even though some dinosaur DNA can be recovered from fossilized remains or perhaps even in a partially digested form from the digestive tracts of resin embedded-insect specimens, it is still not a complete dinosaur genome.

Another obstacle to overcome in cloning an entire dinosaur is amplifying the amount of dinosaur DNA available. The small amounts of dinosaur DNA recovered from any source must be amplified in quantity to be of value for cloning. A third difficulty is sequencing the DNA. Once the tiny amounts of DNA have been amplified, the sequence of bases in the DNA fragments must be determined. Biotechnologists have a variety of techniques available to determine the sequence of bases in any DNA, but this long and tedious step must be completed before any thought of cloning anything can be considered.

These first three technical obstacles have been addressed in recent years. All three of the procedures described are routinely done in laboratories around in the world. However, there are other problems to solve before a baby dinosaur will escape from an artificial egg while humans observe.

Remember that there are no complete dinosaur chromosomes. This means there will be no complete dinosaur genomes recovered so any missing sequences in the dinosaur DNA will have to be "guessed at" by scientists. Of course, there are certain clues as to what might go where to fill in missing spaces in the dinosaur chromosomes, but the truth is, no one knows what complete dinosaur DNA would be. This means that scientists must use trial-and-error in determining what genes go where, or, in fact, what genes should be included at all.

If we assume that scientists could actually discover what genes complete one set of dinosaur chromosomes, there will still be more problems to solve. How do you take the Dino DNA you've just created and make a dinosaur from it? DNA by itself is just a chemical! Scientists have discovered that for some species, specifically amphibians, if you enucleate (remove the nucleus from) a newly fertilized amphibian egg, you can insert a nucleus from another fertilized amphibian egg. In most cases, the new nucleus takes over the regular functions of the cell from which the nucleus was removed. This means what you get after growth of the embryo is an amphibian that had originated in the egg from which the transplanted nucleus was taken. The egg of one amphibian species produced another species because you changed the DNA!

Intellectually, we can extrapolate from removing the nucleus form an egg and transplanting it into another egg to removing the nucleus from a living organism, putting it into an egg, and growing it into a new organism. This new organism would be a clone (an exact copy) of the organism whose nucleus was taken. Since a clone is genetically identical to the animal from which it was cloned, the new organism would be a duplicate of that animal.

The idea of placing dinosaur DNA in an enucleated amphibian egg and growing up something like the Compsognathus in the opening scenario may be good science fiction, but it is fiction. No animal has ever been cloned. The technology to do so may be available, but the particulars to make it practical are lacking.

In the laboratory that follows, you will be given some DNA, which needs to be cut so a specific gene can be inserted into that sequence. The simulation is similar to the concept of repairing fragmented pre-historic DNA with genes, which are considered to be necessary to replace genes missing from the pre-historic DNA.

The simulated dino DNA will be cut with a restriction enzyme, a special enzyme that acts like a molecular scissors and cuts through double-stranded DNA. There are many different restriction enzymes. Some leave a flat or blunt end when they cut. The restriction enzyme you will be using, BamH1, leaves overhanging ends.

The next step involves ligation of the DNA you cut with BamH1. The Simulated Amphibian DNA has already been cut with BamH1, so it already had compatible overhangs, or what molecualr biologists call "sticky ends". They are referred to as sticky because the complementary bases on the overhangs will form hydrogen bonds and help hold the pieces of DNA close together. A different enzyme, T4 Ligase is used to seal pieces of DNA together by making the covalent sugar-phosphate bond in the DNA backbone. After you perform your ligation, the DNA will be larger. To see this, you will run samples of your DNA out on an agarose gel, which separates DNA by size using an electric field. The piece of dino DNA you want to ligate is approximately 2300 base pairs. The piece of amphibian DNA you will be using as filler is approximately 3300 base pairs. The ligated product you will be looking for will be a combination of these two sizes, or 5800 base pairs. You will be able to estimate the size of your products using the Lambda Hind III Size marker. This is a pre-cut piece of DNA where the sizes of each piece are known. By comparing the bands you see in the gel with your samples to the bands of the size marker, you'll be able to estimate the size of your DNA bands.

Be sure to read the entire protocol before you begin.

 

Material e reagentes

Sim. Dino DNA (DNA de dinossáurio, simulado)

Sim. Amphib DNA (DNA de anfíbio, simulado)

Bam HI (enzima de restrição)

Universal Buffer (tampão para Bam HI)

T4 ligase

Ligase Buffer

Loading dye (mistura de amostras para electroforese)

Agarose

TBE (tampão de electroforese)

Stratbloo (corante de DNA)

Lambda Ladder (marcadores de peso molecular)

Microcentrífuga

Banho de água

Microtubos

Microcapilares

Equipamento para electroforese

 

Procedimento experimental Dia 1

1. Distribuir por grupos

2. Responder às questões # 1 e # 2

3. Observar o equipamento disposto na bancada e no laboratório

4. Responder à questão # 3

5. Recolher os materiais para o grupo. MANTER OS TUBOS SEMPRE NO GELO

6. Treinar o uso do microcapilar pipetando volumes de água destilada

7. Marcar um microtubo com a identificação do grupo de lado e CUT na tampa

8. Pipetar 10 ul de Sim. Dino DNA para o fundo do microtubo

9. Lavar o microcapilar com água destilada

10. Adicionar9 ul de Universal buffer sem tocar no líquido existente

11. Lavar o microcapilar com água destilada

12. Adicional 1 ul de Bam HI sem tocar no líquido existente

13. Remover o tubo do gelo, tapar e misturar o conteúdo

14. Centrifugar durante 3 segundos

15. Incubar no banho de água a 37C durante 45 minutos

16. Responder às questões # 4 e # 5

17. Retirar o microtubo do banho e arrefecer no gelo

18. Verificar a marcação do tubo e guardar para continuação do trabalho

 

Procedimento experimental Dia 2

1. Recuperar o microtubo da aula anterior e o restante material necessário

2. Marcar um microtubo novo com a identificação do grupo de lado e LIG+ na tampa

3. Pipetar 10 ul do DNA digerido na aula anterior (CUT) para o novo microtubo (LIG+)

4. Lavar o microcapilar com água destilada

5. Adicionar 10 ul de Sim. Amphib DNA

6. Lavar o microcapilar com água destilada

7. Adicionar 2 ul de Ligase Buffer

8. Lavar o microcapilar com água destilada

9. Adicionar 1 ul de T4 ligase

10. Lavar o microcapilar com água destilada

11. Remover o tubo do gelo, tapar e misturar o conteúdo

12. Centrifugar durante 3 segundos

13. Incubar à temperatura ambiente durante 30 minutos

14. Responder às questões # 6 e # 7

15. Após a incubação adicionar 2 ul de Loading dye em cada microtubo (CUT e LIG+)

16. Lavar o microcapilar com água destilada

17. Verificar a marcação dos tubos e guardar para continuação do trabalho

 

Procedimento experimental Dia 3

1. Recuperar os microtubos da aula anterior e o restante material necessário

2. Observar o gel de agarose (a partilhar com outros grupos)

3. Identificar os poços com desenho no caderno do laboratório

4. Pipetar 9-10 ul do tubo CUT. Depositar cuidadosamente no fundo do poço do gel, começando pelo lado esquerdo.

5. Lavar o microcapilar com água destilada

6. Pipetar 9-10 ul do tubo LIG+. Depositar no poço seguinte. Anotar

7. Lavar o microcapilar com água destilada

8. Pipetar 9-10 ul de Sim. Amphib DNA. Depositar no poço seguinte. Anotar

9. Lavar o microcapilar com água destilada

10. Pipetar 9-10 ul de Sim. Dino DNA. Depositar no poço seguinte. Anotar

11. Lavar o microcapilar com água destilada

12. Pipetar 9-10 ul de Lambda Ladder. Depositar no poço seguinte. Anotar

13. Lavar o microcapilar com água destilada

14. Preparar o sistema de electroforese com ligação à fonte de alimentação

15. Aplicar uma corrente de 70-110 volts até a frente se posicionar a 1-2 cm da extremidade do gel (cerca de 40 minutos)

16. Responder às questões # 8 e # 9

17. Desligar a corrente eléctrica e remover o gel da tina

18. Observar as bandas e registar as observações

19. Responder à questões #10

20. Responder, depois da aula, às questões # 11 e # 12

 

Procedimento experimental Dia 4

Análise das respostas de cada grupo às questões

Avaliação crítica do trabalho realizado

 

QUESTÕES

1. Porque razão é necessário manter no gelo as soluções de DNA e de enzima?

2. Descreva as funções enzimáticas da enzima de restrição e da ligase.

3. Onde está a enzima de restrição que vai usar?

4. Como é que o DNA do dinossáurio poderia ser amplificado depois de isolado?

5. Quais dos problemas descritos na introdução considera mais severos? Qual o problema mais fácil de resolver? Justifique a sua resposta com 3 ou 4 frases.

6. Que é um clone? Explique como procederia para clonar uma rã.

7. Escreva uma hipótese do que sucederá quando junta no mesmo tubo o DNA de dinossáurio, o DNA de anfíbio e a ligase.

8. Porque é que é importante registar qual a amostra de cada tubo que foi aplicada em cada poço?

9. Que julga que acontece ao DNA durante a electroforese. Não use mais de 3 ou 4 frases.

10. Desenhe uma imagem do seu gel no caderno do laboratório. Assegure-se que todos os poços estão devidamente identificados.

11. No desenho anterior indique a banda do DNA que esperava obter após a realização do trabalho. Justifique

12. Continue a história do cenário, tentando descrever um final feliz para a descrição que consta do cenário.

sumário

"Transformação Bacteriana" (Trabalho adaptado de Kit educacional da Stratagene)

Student Information and Instructions

Primary Objective

Perform and evaluate a transformation of bacterial cells.

Other Objectives

1. Define: genetics, recombinant DNA, plasmid, Luciferase.

2. Compare the functions of restriction endonucleases and DNA ligases.

3. Explain and evaluate the function and importance of DNA.

4. Review the functions of proteins in cells.

5. Compare and contrast three prokaryotic procedures for passing DNA from one cell to another.

6. Employ genetic engineering techniques of plasmid manipulation to prepare a bacterial transformation of Escherichia coli. Examine and evaluate your results

7. Calculate the transformation efficiency of your experimental protocol.

 

Scenario

Jerry's mother came back from taking Todd to the doctor. She carried him easily in her arms, although at a year and a half he should have been twice the size. She sat down on the couch next to Jerry.

"Jerry, You know Todd should be bigger, and the doctor has been doing tests on him to see what's wrong. Well, we got the results."

"So, is he okay? Is it serious?" Jerry asked.

"Well, it could be serious, but we caught it in time. Todd's body doesn't make enough growth hormone. So the doctor says we'll have to give him this hormone periodically as he grows up. With proper treatment, he'll be okay."

Jerry sighed with relief. He'd been very worried about his little brother.

"The doctor also said we are very lucky. A few years ago, there wouldn't have been enough growth hormone available to treat Todd, but now that they've cloned it, there's plenty of medicine for him."

"What do you mean, cloned it?"

Jerry's mother looked puzzled. "You know, I don't know what that means. I'll ask next time I'm at the doctor's."

^^^^^^^^^^^^^^^^^^^^^^^^^^^

The next day at school, Jerry waited around after his biology class. His teacher, Ms. Anderson, looked up.

"What's up, Jerry?"

Jerry explained about Todd and his medication. "So what does cloned mean?"

Ms. Anderson grabbed a notepad and a pen. "You know that we pass traits on from one generation to the next through genes. These genes are located in a molecule called deoxyribonucleic acid (DNA) in our cells. The genes code for proteins, and what proteins a cell makes controls what it looks like and what it can do."

"Yeah, so... what's that got to do with cloning?", Jerry was beginning to be sorry he'd asked. He didn't expect a review of the year's biology class.

"Well,", Ms. Anderson continued, "scientists know that the protein codes in DNA are universal, meaning all living things have virtually the same code. So what the scientists have learned to do is take the gene for a particular protein from one organism and put it into another."

"They're gonna put a gene into my brother?" Jerry was upset.

"Oh, no, Jerry. Nothing like that. Let me explain some more." Ms. Anderson continued. "What they've done in your brother's case is take the gene for human growth hormone, put it into a small piece of DNA called a plasmid, and then put it into bacteria. The bacteria then make the growth hormone and the scientists can collect and purify it."

"They're gonna give my brother bacteria's growth hormone? Is he gonna grown into a germ?"

Ms. Anderson laughed. "No, not at all - remember the code to make protein is universal. The bacterial makes human protein because the human gene is put into the bacteria."

"How do they get it in there?"

"Well, I was just about to set up an experiment about that - it's called bacterial transformation - want to see?"

"Okay"

They walked to the back of the classroom where Jerry saw beakers of ice, bacterial plates, test tubes, and a waterbath.

"Bacteria have a cell wall that protects them from the environment, and would keep out any DNA we tried to get in. We can 'poke' small holes in the cell wall by treating them with a chemical. When we do this we say the bacteria are competent.

Ms. Anderson picked up a test tube. "The bacteria that are growing in the media in this test tube are sensitive to the antibiotic ampicillin. Our experiment is to try to give them the ability to resist ampicillin."

Ms. Anderson held up 2 test tubes on ice. "These are the same bacteria, but now they are competent. I've treated them with calcium chloride. This helps put holes in their walls." She put the tubes back on ice and handed Jerry a tube with a small amount of clear liquid in the bottom. "This is the plasmid that contains the gene for ampicillin resistance - It's the DNA we want to get into the bacteria." She handed Jerry a pipettor, "now take some of the plasmid and put it into one tube and mark that tube AMP+. Mark the other tube AMP-."

Jerry put the plasmid into the bacteria and they put both tubes back on ice.

"Why do you have two tubes?"

"One's the control. You remember what a control is?" Ms. Anderson asked.

"Yes, it's to show the changes in the experiment are really due to what you do."

"Yes, basically, that's the idea. The bacteria now have to be heat-shocked - we put them at a hotter temperature for a short period of time. That helps them take up the DNA. Then we put them back on ice for a few minutes."

After letting the bacteria sit on ice a few minutes, Jerry and Ms. Anderson put some of the bacteria on plates and spread them out. "We spread them out so all the bacteria don't grow in the same place." Ms. Anderson said.

"How come there are 2 different piles of plates?"

"These plates, Jerry, have ampicillin in them, these plates don't. Let's see if you can make a hypothesis about what is going to happen."

What do you expect if I put bacteria from the Amp+ tube on an Ampicillin Plate? Will it grow? What if I put bacteria from the AMP- tube on an ampicillin plate? Will that grow?

Jerry and Ms. Anderson talked about the possibilities for a while.

"So, how does this relate to cloning the gene for my brother's medicine?" Jerry asked.

"Well, after the scientists identify the gene they want, they cut open a plasmid, put the gene in the plasmid, and then put the plasmid into bacteria by transforming them." Do you understand?"

"Yes, I think so. Can I come back tomorrow and see which bacteria grew?"

Ms. Anderson said yes and Jerry ran home to explain to his mother that bacteria made his brother's medicine.

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Introduction

Genetics is the study of how inheritable traits are passed from one generation to the next. Traits are the physical expression of genes coded for by deoxyribonucleic acid (DNA). DNA transmits patterns for making proteins in a cell. The phenotype of a cell is the collection of the proteins expressed in that cell. Some of these proteins are enzymes, other proteins are structural, for example serving as part of the cellular membrane or as a membrane receptor. Among their many functions, various proteins determine the structure of a cell and which reactions will occur in each cell. It is the collection of proteins within a living cell that makes that cell unique.

To study genetics, scientists can spend many generations observing and recording inheritable human traits or spend years studying plants and their seedlings. Often they choose to gather genetic information from bacteria because they can collect information in a few days. The most common bacteria used in laboratories is the strain Escherichia coli. Bacteria reproduce a new generation every 20-30 minutes under optimal conditions. Because of the short reproductive time and the ease of growth, bacterial genetics is an excellent way to begin studying inheritable traits.

Bacterial DNA Transfer

Most bacteria simply divide to form the next generation. In this asexual form of reproduction, each daughter cell receives a nearly identical copy of the single parent cell's DNA. Binary fission is a quick and easy way to reproduce but with little genetic diversity.

Occasionally, bacterial cells use one of three prokaryotic procedures to combine DNA from different sources: This leads to a new generation that is genetically different from either parent. When any of the three processes occurs, DNA from one cell enters a bacteria. From within that second cell, the newly combined nucleic acid can pass on to the next generation and the next. This redistribution of DNA assures genetic diversity.

The three DNA exchange processes include:

conjugation, in which DNA passes from a donor bacterial cell to a recipient bacteria, This reproduction requires a specialized cell structure called a pilus which physically connects the two cells and acts as a passageway for DNA material,

transduction occurs when an existing virus infecting a bacteria takes a small piece of bacterial host DNA and carries it, along with its own DNA, into the next bacteria it infects,

transformation, the third type of DNA exchange, when naked bits of foreign DNA enter competent (receptive) bacterial cells. Interestingly, the foreign DNA entering the competent bacteria does not have to be bacterial; - plants, animals, and fungi all can contribute DNA to the next bacterial generation.

Bacterial Transformation

Scientists in biotechnology move DNA from one genus into another in their laboratories, creating recombinant DNA. Bacteria carrying recombinant DNA now supply us with human insulin, human growth hormone, bovine growth hormone, and other useful products.

In the laboratory, geneticists insert intact plasmids (small, extrachromosomal circles of DNA) into various microorganisms. Since the plasmids make exact copies (clones) of themselves and are used as carriers for foreign DNA, the plasmids are called cloning vectors.

Molecular biologists cut open plasmids with restriction endonucleases (enzymes that cleave DNA). They then attach foreign DNA in the opening and reseal the cloning vector with another enzyme, DNA ligase, in a process called ligation.

In this exercise, you perform two different bacterial transformations of Escherichia coli, one with the pUC18 plasmid which carries a gene for resistance to the antibiotic ampicillin, the second with the plasmid pHotobac*, which carries not only the ampicillin-resistance gene but also the bioluminescent operon from a Photobacterium species, a marine microorganism. Genetic transformation occurs when a cell takes up a piece of genetic material and expresses a gene contained on the DNA. This gene product (usually a protein) can alter the phenotype of the organism.

What is bioluminescence? It is the biochemical conversion of chemical energy into light energy. Many organisms are capable of bioluminescence: fireflies, fungi, fish, squid, clams, and bacteria. Many marine organisms contain bacterial symbionts that provide bioluminescence. The firefly is an example of an organism which does not require symbionts

First you suspend ampicillin sensitive Escherichia coli (E.coli Amp-) cells in three tubes of chilled calcium chloride (CaCl2) solution. To one of the tubes, you add pUC18, the plasmid, with its ampicillin resistance gene. To the second tube, you'll add pHotobac*, the plasmid with the Luciferase operon and ampicillin-resistance gene. To the third tube you add nothing (the control). After a short incubation, you heat shock the tubes of E. coli to help the bacteria take up the plasmid. You'll then plate out the bacteria on LB agar plates with and without ampicillin.

If transformation occurs, you will recover colonies of E. coli that are now ampicillin resistant (Amp+). Remember that the original bacterial culture was sensitive to ampicillin. (Amp-), and only the bacterial cells that took up the plasmid containing the Ampicillin resistance gene you added in this experiment will be resistant to the antibiotic. How will you discover whether a cell is ampicillin resistant or not?

To see if transformation took place, you inoculate, incubate, and then examine ampicillin-containing agar plates spread with bacteria from both tubes. Why? You also inspect inoculated, ampicillin-free plates; will there be growth? Why do you add pUC18 to only one of the tubes, not both?

In addition to altering the phenotype of the bacteria for ampicillin resistance, the bacteria transformed with pHotobac* will also show the bioluminescence of the Luciferase operon. Why won't the AMP+ bacteria transformed with pUC18 show bioluminescence?

Genetic engineers need to compare the effectiveness of various transformation protocols, so they calculate a procedure's transformation efficiency. The transformation efficiency is the number of transformants (in this case, E. coli Amp+) per microgram of plasmid used in an experiment. You will calculate the transformation efficiency of your work in this exercise.

 

PROCEDURE for Bacterial Transformation Day 1

1. Label 3 sterile 1.5 ml tubes, one is "pUC" , one "pHotobac", and the other is "control".

2. Using sterile technique, transfer 250 ul (5 drops) of transformation solution (calcium chloride) into each tube. Keep all tubes on ice.

3. Using a sterile inoculating loop, run the loop along a bacterial streak on the Xl-1 plate. Resuspend the bacteria in the transformation solution by rapidly swirling the end of the loop in the solution. Do this for all tubes. It is important that the cells be dispersed through the liquid.

4. Place on ice for 15 minutes.

5. Use a sterile loop to transfer 10 ul of 0.001ug/ul pUC18 (plasmid DNA) solution into your "pUC" tube. Cap and mix briefly by tapping tube against your fingers to mix the DNA into your bacterial suspension.

6. Use a sterile loop to transfer 10 ul of 0.001ug/ul pHotobac* (plasmid DNA) solution into your "pHotobac" tube. Cap and mix briefly by tapping tube against your fingers to mix the DNA into your bacterial suspension.

7.Incubate your closed tubes iced for 20-30 minutes.

8. While the tubes are incubating on ice, prepare your plates:

You'll need six plates: 3 LB (no ampicillin) and 3 LB/AMP (with ampicillin in the agar).

Label the plates (on the bottoms since the tops can be mixed up) as indicated on the following chart. Try to write small, but clearly.

tube LB plate LB/AMP plate

control tube LB control LB/AMP control

pUC 18 pUC LB pUC LB/AMP

pHotobac* pHoto LB pHotoLB/AMP

9 After the incubation on ice, heat shock your tubes of bacteria by removing them from the ice and immediately placing them in a 42o C waterbath for 45 seconds.

10. Return the tubes to ice for 2 minutes.

11. Using sterile technique, pipette 250ul LB Broth (5 drops) into each tube. Mix each tube's contents by gently tapping each closed tube with your finger.

12. Allow the bacteria to incubate at 37oC for 15-20 minutes This allows the antibiotic resistance gene to express.

13. Employing sterile technique, place a sterile innoculating loop into the control tube. Spread the bacteria onto the LB control plate using the loop. Still using sterile technique, place the loop back into the control tube. Spread the bacteria onto the LB/AMP control plate using the loop. Dispose of the loop in a bleach solution.

14. Do the same for the tubes containing the pUC transformation. Employing sterile technique, place a sterile innoculating loop into the pUC tube. Spread the bacteria onto the pUC LB plate using the loop. Still using sterile technique, place the loop back into the pUC tube. Spread the bacteria onto the pUC LB/AMP plate using the loop. Dispose of the loop in a bleach solution.

15.Do the same for the tube containing the pHotobac* transformation. Employing sterile technique, place a sterile innoculating loop into the pHotobac tube. Spread the bacteria onto the pHotobac LB plate using the loop. Still using sterile technique, place the loop back into the pHotobac tube. Spread the bacteria onto the pHotobac LB/AMP plate using the loop. Dispose of the loop in a bleach solution.

16. Wait a few minutes before inverting the plates and incubating them at 37oC for 24 hours.

17. Calculate and record how much pUC18 and how much pHotobac* you transferred in step (4).

 

PROCEDURE for Bacterial Transformation Day 2

1. Examine microbial growth on each of the 6 Petri plates in your bacterial transformation experiment. Count colonies on countable plates-If there are so many colonies you can't see individual colonies, label those TMTC (too many to count). Which plates have colonies that are too numerous to count ?

2. Compare and contrast growth on the Petri dishes and explain the results that you observe. Do you get growth on all plates? Why do the bacteria with the ampicillin plasmid grow on plain LB plates? Why don't bacteria without the ampicillin plasmid grow on plates containing ampicillin? Which plates are controls? Do both the positive and negative control plates have the same number of colonies? Are there fewer colonies on the pUC/ LB AMP plate than on the control LB AMP plate? Why?

3. Calculate the transformation efficiency of this procedure. (This is the number of Amp+ colonies per microgram of pUC18 plasmid DNA or the number of colonies of pHotobac* per microgram of plasmid DNA).

4. Discuss factors that might affect the transformation efficiency of a plasmid suspension. What could you do to produce more Amp resistance colonies?

5. Take the plates to a dark room. Examine the plates (They should be 24-48 hours old -sooner and the luciferase may not have had time to work, older and the luciferase may have broken down). Which plates have glowing colonies? Are all the colonies on the pHotobac plates glowing? Are any of the colonies on the pUC plates glowing? Does it matter if the transformed bacteria are on LB or LB/AMP plates?

 

Name ____________________ Section:____________________

 

Laboratory Report for Bacterial Transformation

1. Discuss the function of each plate in the design of this bacterial transformation experiment. Why did you need to include each control, and which plate(s) were the "experimental plate"?

LB control

LB/AMP control

pUC LB

pUC LB/AMP

pHoto LB

pHotoLB/AMP

2. Use the following formula to calculate the mass in ug of pUC18 that you transferred in step (4):

Concentration of plasmid

X

Volume of plasmid used

=

total amount used

___ug/ul

X

____ul

=

____ug of pUC18 transferred

3. Gather data from your transformation experiment by examining any Escherichia coli growth on your Petri plates. Count colonies on countable plates. Record TMTC for plates with too many colonies to count. Record 0 for plates that show no Escherichia coli growth. You may use control plates from other groups to complete your data.

LB plates # of colonies LB/AMP plates # of colonies

LB control LB/AMP control

pUC LB pUC LB/AMP

pHoto LB pHotoLB/AMP

4. Compare and contrast growth/ bioluminescence on each of the sets of plates listed and explain the results that you observe:

LB control and LB/amp control -

pUC LB and pUC LB/AMP -

pHotobac* LB and pHotobac* LB/AMP -

LB/AMP control and pUC LB/AMP -

LB/AMP control and pHotobac* LB/AMP -

pUC plates and pHotobac* plates -

5. Calculate the transformation efficiency of this experimental protocol by determining how much pUC18 you employed to produce the number of transformants (Amp+ colonies) that you observed.

A. What was the total amount in ug of pUC18 that you added to your bacterial cell suspension?

We need to calculate the amount of DNA added to the tube. You added 10 ul of .001 ug/ul pUC to the tube. To calculate the amount of DNA in the tube: (remember to watch your units!)

10ul X (0.001 ug/ul) = 0.01 ug plasmid DNA added to the tube.

B. Use the following equation to calculate approximately what fraction of the volume of your cell suspension you spread on the pUC LB/amp plate:

Volume of suspension spread /Total volume of suspension = fraction spread

The total volume consisted of the transformation solution and the LB broth

250ul (Step 1) + 250 ul (step 11) = 500ul total volume

Since you spread 10ul (step 14) of the volume of the pUC- transformed bacteria, the fraction you spread was:

10ul/500ul= .02 or 2%

C. Use the following equation to calculate the mass of the pUC18 in the bacterial suspension that you spread onto your +AMP plate:

Total amount of pUC18 X fraction spread = amount of pUC18 spread

First, we need the amount of DNA added to the tube, calculated in Step A

Now we calculate how much of that DNA was in the fraction of the bacteria that you spread on the plate:

0.01ug/tube X 0.02 (fraction spread on plate) = 0.0002 ug of pUC18 spread

D. If possible, calculate the transformation efficiency of your experiment using the following equation to determine the number of transformant colonies that developed per ug of pUC18.

# of colonies on pUC LB/AMP plate / mass of pUC18 spread = transformation efficiency

___colonies / .0002ug = transformation efficiency

6. Calculate the transformation efficiency for pHotobac* using the same procedures described in step 5.

7. Discuss experimental factors that might influence the transformation efficiency of a plasmid suspension.

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