BISC220/S14: Mod 1 Lab 2: Difference between revisions

From OpenWetWare
Jump to navigationJump to search
 
(19 intermediate revisions by the same user not shown)
Line 12: Line 12:
</div>
</div>
== Enzyme Structure/Function: Background Information ==
== Enzyme Structure/Function: Background Information ==
The purification of an enzyme is often required in order to study its physical characteristics and the reaction it catalyzes. Purification is essentially a concentrating process in which the concentration of the target enzyme is increased while other proteins are eliminated. The enrichment process also eliminates metabolites and other cellular constituents that may influence the enzyme structure and/or the reaction. Background information on protein purification can be found below and also in your textbook.<br>
The purification of an enzyme is often required in order to study its physical characteristics and the reaction it catalyzes. Purification is essentially a concentrating process in which the concentration of the target enzyme is increased while other proteins are eliminated. The enrichment process also removes metabolites and other cellular constituents that may influence the enzyme structure and/or the reaction. Background information on protein purification can be found below and also in your textbook.<br>
<br>
<br>
During our first lab series, we will be partially purifying and characterizing the enzyme β-galactosidase from ''Escherichia coli'', strain BL21(pET-14b).  Although we will not be concerned with regulation of gene expression, it is of interest that experimentation on the production of β-galactosidase by ''E. coli'' ultimately led Francois Jacob and Jacques Monod to propose the 'operon' model for the regulation of gene expression. If you are interested in learning more about transcription regulation and early work on the genetic analysis of the lac-operon in ''E. coli'', your textbook is a good resource.  <br>
During our first lab series, we will be partially purifying and characterizing the enzyme β-galactosidase from ''Escherichia coli'', strain BL21(pET-14b).  Although we will not be concerned with regulation of gene expression, it is of interest that experimentation on the production of β-galactosidase by ''E. coli'' ultimately led Francois Jacob and Jacques Monod to propose the 'operon' model for the regulation of gene expression. If you are interested in learning more about transcription regulation and early work on the genetic analysis of the lac-operon in ''E. coli'', your textbook is a good resource.  <br>
<br>
<br>
To study protein structure and function, we have chosen the enzyme β-galactosidase for a variety of reasons. First, it is a convenient enzyme for learning basic techniques in enzymology because it is readily available in large quantities from microbial cells and is relatively stable. Next, the gene has been fully sequenced and cloned. The crystal structure of the protein and hence its 3-D configuration has been solved. Finally, the specific activity assay generally used to assess enzyme function is reliable and convenient (Jacobson et. al., 1994; Ring, 1990; Wallenfels and Weil, 1972).<br>
To study protein structure and function, we have chosen the enzyme β-galactosidase for a variety of reasons. First, it is a convenient enzyme for learning basic techniques in enzymology because it is readily available in large quantities from microbial cells and is relatively stable. Next, the gene has been fully sequenced and cloned. The crystal structure of the protein and hence its 3-D configuration has been solved. Finally, the specific activity assay generally used to assess enzyme function is reliable and convenient (Jacobson et al., 1994; Ring, 1990; Wallenfels and Weil, 1972).<br>
<br>
<br>
Βeta-galactosidase allows ''E. coli'' to grow in a culture medium containing the sugar lactose as the sole source of energy. The enzyme hydrolyzes lactose into glucose and galactose:<br>
Βeta-galactosidase allows ''E. coli'' to grow in a culture medium containing the sugar lactose as the sole source of energy. The enzyme catalyzes the hydrolysis of lactose into glucose and galactose:<br>
<br>
<br>
<center>[[Image:Lactose bgal image.jpg]]</center>
<center>[[Image:Lactose bgal image.jpg]]</center>
<br>
<br>
The purification of β-galactosidase is facilitated when ''E. coli'' produce large quantities of the enzyme. Growth on lactose as a carbon source is not a sufficient means of insuring abundant production of the enzyme since the expression of β-galactosidase is controlled. β-galactosidase production remains partially repressed even when cells are grown in the presence of the substrate lactose. It is possible, however, to transform genetically engineered strains of ''E. coli'' with a plasmid that expresses high levels of β-galactosidase. The plasmid fuses a strong promoter upstream of the β-galactosidase gene, ''lacZ''. The gene promoter (and the gene’s over expression) is regulated by isopropyl-β-D-thiogalactoside (IPTG), the structure of which is shown below. <br>
The purification of β-galactosidase is facilitated when ''E. coli'' produce large quantities of the enzyme. Growth on lactose as a carbon source is not a sufficient means of insuring abundant production of the enzyme since the expression of β-galactosidase is controlled. β-galactosidase production remains partially repressed even when cells are grown in the presence of the substrate lactose. It is possible, however, to transform genetically engineered strains of ''E. coli'' with a plasmid that expresses high levels of β-galactosidase. The plasmid fuses a strong promoter upstream of the β-galactosidase gene, ''lacZ''. The gene promoter (and overexpression of the gene product) is regulated by isopropyl-β-D-thiogalactoside (IPTG), the structure of which is shown below. <br>
<br>
<br>
<center>[[Image:IPTG image.jpg]]</center><br>
<center>[[Image:IPTG image.jpg]]</center><br>
<br>
<br>
In addition to allowing the production of large quantities of β-galactosidase, the ''lacZ'' gene on the plasmid, is modified so that the primary sequence of the enzyme is extended on the amino terminus with six histidines. This 6xHis tail can be exploited to purify the protein using metal chelate affinity chromatography (Kagedal, 1998).  See Lab 2 for additional background information on metal chelate affinity chromatography. <br>
In addition to allowing the production of large quantities of β-galactosidase, the ''lacZ'' gene on the plasmid, is modified so that the primary sequence of the enzyme is extended on the amino terminus with six histidines. This 6xHis tag can be exploited to purify the protein using metal chelate affinity chromatography (Kagedal, 1998), which we will perform in Lab 2.<br>
<br>
<br>
'''Our goals for Lab 1 are:'''<br>
'''Our goals for Lab 1 are:'''<br>
<br>
# Calibrate our micropipets
# Calibrate our micropipets,
# Induce ''E. coli'' BL21(pET-14b) cells to produce large quantities of 6xHis tagged β-galactosidase  
# Induce ''E. coli'' BL21(pET-14b) cells to produce large quantities of 6xHis tagged β-galactosidase, and
# Learn how to model proteins and analyze their sequences, using the computer programs RasMol and [http://www.ebi.ac.uk/Tools/msa/clustalo/ Clustal Omega].<br>
# Learn how to model macromolecules and analyze their sequences, using the computer programs RasMol and [http://www.ebi.ac.uk/clustalw/# ClustalW].<br>
<br>
<br>
<br>
<br>
'''References:'''<br>
'''References:'''<br>
<br>
 
Jacobson RH, Zhang XJ, DuBose RF, Matthews BW (1994) Three-dimensional structure of β-galactosidase from ''E. coli''. ''Nature''. 369:761-6.<br>
Jacobson, R.H., Zhang, X.J., DuBose, R.F., and Matthews, B.W. (1994). Three-dimensional structure of β-galactosidase from ''E. coli''. Nature ''369,'' 761-6.<br>
<br>
 
Karp G (2005) ''Cell and Molecular Biology Concepts and Experiments, 4th edition'', John Wiley & Sons, Inc.,Hoboken. <br>
Karp, G. (2005). Cell and Molecular Biology Concepts and Experiments, 4th edition (Hoboken: John Wiley & Sons, Inc.). <br>
<br>
 
Ring M, Huber RE (1990) Multiple replacements establish the importance of tyrosine-503 in beta-galactosidase (''Escherichia coli''). ''Arch Biochem Biophys''. 283:342-50.<br>
Ring, M., Huber, R.E. (1990). Multiple replacements establish the importance of tyrosine-503 in beta-galactosidase (''Escherichia coli''). Arch Biochem Biophys ''283,'' 342-50.<br>
<br>
 
Wallenfels K, Weil R (1972) “β-galactosidase” In ''The Enzymes'' (Boyer PD, ed), Vol VII, 3rd ed, Academic Press, New York, pp 617-663.<br>
Wallenfels, K. and Weil, R. (1972). β-galactosidase. In The Enzymes, Vol VII, 3rd ed PD Boyer, ed. (New York: Academic Press), pp. 617-663.
<br>
<br>
<br>
<br>
Line 50: Line 49:


[[BISC220/S14: Mod 1 RasMol Tutorial| RasMol Tutorial]] <br>
[[BISC220/S14: Mod 1 RasMol Tutorial| RasMol Tutorial]] <br>
[[BISC220/S14: Mod 1 ClustalW Tutorial | ClustalW Tutorial]]<br>
[[BISC220/S14: Mod 1 Clustal Omega Tutorial | Clustal Omega Tutorial]]<br>
<br>
<br>
<br>


Line 57: Line 55:
'''THE INDUCTION OF β-GALACTOSIDASE'''<br>
'''THE INDUCTION OF β-GALACTOSIDASE'''<br>
<br>
<br>
You will be provided with approximately 200 ml of ''E. coli'' BL21 (pET-14b) in log phase growth in Luria broth (LB) media. You will induce the cells of this genetically engineered strain to increase production of 6xHis-β-galactosidase by adding IPTG to the cells and incubating them at 37°C for one hour. You will collect the cells by centrifugation and freeze the cell pellet until the next lab period.<br>
You will be provided with approximately 200 mL of ''E. coli'' BL21 (pET-14b) in log phase growth in Luria broth (LB) media. You will induce this genetically engineered strain to increase production of 6xHis-β-galactosidase by adding IPTG to the cells and incubating them at 37°C for one hour. You will collect the cells by centrifugation and freeze the cell pellet until the next lab period.<br>
<br>
# Remove a flask containing the ''E. coli'' BL21(pET-14) cells from the 37°C shaking incubator, and bring it to your lab bench.
# Remove a flask containing the ''E. coli'' BL21(pET-14) cells from the 37°C shaking incubator and bring it to your lab bench.
# Using a sterile 1 mL pipette and aseptic technique, remove 1.0 mL of the culture and add it to a microfuge tube labeled "pre-IPTG cells" on tape of your team color. Please also include the initials of your group and lab day. Centrifuge this small aliquot of cells for 2 minutes in a microcentrifuge.  Discard the supernatant into the waste container next to the centrifuge and give the labeled dry pellet to your instructor to freeze at -80°C.  
# Using a sterile 1 ml pipette and aseptic technique, remove 1.0 ml of the culture and add it to a microfuge tube labeled "pre-IPTG cells" on tape of your team color. Please also include the initials of your group. Centrifuge this small aliquot of cells for 2 minutes in a microcentrifuge.  Discard the supernatant into the waste container next to the centrifuge and give the labeled dry pellet to your instructor to freeze at -70°C.  
#Using a sterile 5.0 mL pipette and aseptic technique, add 2.0 mL of 0.1M IPTG to the flask of growing ''E. coli'' cells.  This addition will yield a final concentration of approximately 1.0 mM IPTG in the culture flask.
#Using a sterile 5.0 ml pipette and aseptic technique, add 2.0 ml of 0.1M IPTG to the flask of growing ''E. coli'' cells.  This addition will yield a final concentration of approximately 1.0 mM IPTG in the culture flask.
#Return the flask of ''E. coli'' cells containing IPTG to the incubator and allow the cells to incubate with shaking for one hour.  During this time IPTG will induce production of large amounts of β-galactosidase.  
#Return the flask of ''E. coli'' cells containing IPTG to the incubator and allow the cells to incubate with shaking for 60 minutes.  During this time IPTG will induce production of large amounts of β-galactosidase.  
#Calibrate your micropipettes while your protein is synthesized by our genetically engineered strain of ''E. coli''. Your instructor will demonstrate how to use the micropipets. Then follow the calibration protocol below.  
#Calibrate your micropipets while your protein is synthesized by our genetically engineered strain of ''E. coli''. Your instructor will demonstrate how to use the micropipets. Then follow the calibration protocol on the next page.  
#After the incubation period, remove the flask of ''E. coli'' from the incubator. Remove 1.0 mL of the culture to a microcentrifuge tube labeled "post-IPTG cells". Make sure the label includes the initials of your lab group, team color, & lab day.  Pellet the cells as in step 2 above. Give the labeled tube containing the dry pellet to your instructor for freezing at -80°C.
#After the incubation period, remove the flask of bacterial cells from the incubator. Remove 1.0 ml of the culture to a microfuge tube labeled "+ IPTG cells". Make sure the label includes the initials of your lab group, team color, & lab day.  Pellet the cells as in step 2 above. Give the labeled tube containing the dry pellet to your instructor for freezing at -70°C.
#Pour the post-induction cells into an empty centrifuge bottle with a screw-cap provided by your lab instructor; secure the cap.  Do not overfill the centrifuge bottle! If you have extra culture, it should be left in the flask to be autoclaved before disposal. Please do not throw any bacterial cells down the drain!
#Pour the post-induction cells into an empty centrifuge bottle with a screw-cap provided by your lab instructor; secure the cap.  Do not overfill the centrifuge bottle. If you have extra culture, it should be left in the flask to be autoclaved before disposal. Please do not throw any bacterial cells down the drain!
#Centrifuge the cells for 15 minutes at 8,000 RPM (~10,400x g) in a big Sorval centrifuge using the GSA rotor. Be sure that your bottle is balanced with another group’s before starting the centrifuge. Use sterile LB media for equalizing the weights.
#Centrifuge the cells for 15 minutes at 8,000 RPM (~10,400x g) in a big Sorval centrifuge using the GSA rotor. Be sure that your bottle is balanced with another group’s before starting the centrifuge. Use sterile LB media for equalizing the weights.
#Carefully pour the supernatant back into the flask. Remove with a Pasteur pipet any media that remains.  Label the bottle containing your cell pellet with the name of the cells (''E. coli'' BL21(pET-14) + IPTG) on a piece of tape in your team color and include your initials and lab day; give your instructor the bottle to freeze at -70°C until the next lab period.
#Carefully pour the supernatant back into the flask. Remove with a Pasteur pipette any media that remains.  Label the bottle containing your cell pellet with the name of the cells (''E. coli'' BL21(pET-14) + IPTG) on a piece of tape in your team color and include your initials and lab day; give your instructor the bottle to freeze at -80°C until the next lab period.<br>
<br>
For a Word™ format protocol: [[Media:Protocol for the induction of beta-gal.doc‎‎ | Induction of β-gal]]<br>
For a Word™ format protocol: [[Media:PROTOCOL FOR THE INDUCTION OF βgal.doc]]<br>
<br>
<br>
<br>
<br>
'''Protocol for Micropipette Calibration'''<br>
'''Protocol for Micropipette Calibration'''<br>
<br>
#To calibrate your P 1000, P 200, and P 20 micropipettes, label 6 microcentrifuge tubes (1-6) and weigh them. Record the weights in the table below.
#To calibrate your P1000, P 200, and P 20 micropipets, label 6 microfuge tubes (1-6) and weigh them. Record the weights in the table below.
#Following the table below, pipet the specified volumes into the pre-weighed microcentrifuge tubes prepared above and then re-weigh them. Record all weights.
#Following the table below, pipet the specified volumes into the pre-weighed microfuge tubes prepared above and then re-weigh them. Record all weights.
#Calculate the weight of the water in grams. 1000 μL of water should weigh 1 g at room temperature.
#Calculate the weight of the water in grams. 1000 microliters of water should weigh 1 gram at room temperature.
#If the water in any tube weighs more or less than 1 gram, ask your instructor for help. If your calibration is significantly off after several repeated attempts, your pipette (or your technique!) may need adjustment.<br>
#If the water in any tube weighs more or less than 1 gram, ask your instructor for help. If your calibration is significantly off after several repeated attempts, your pipet (or your technique!) may need adjustment.<br>
<br>
<br>
{| border="1"  
{| border="1"  
Line 101: Line 96:
| || <center> 20 (5 times) </center>  || <center> 0 </center> || <center> 900 </center> ||  ||  |
| || <center> 20 (5 times) </center>  || <center> 0 </center> || <center> 900 </center> ||  ||  |
|-
|-
|}
|}<br>
<br>
For a Word™ format protocol: [[Media:BISC220 Protocol for micropipette calibration.doc‎ | Micropipette calibration]]
For a Word™ format protocol: [[Media:Protocol for Micropipet Calibration.doc]]
 
<br>
<br>

Latest revision as of 12:58, 8 July 2013

Wellesley College     BISC 220     Cellular Physiology

Home        Lab Calendars/Point Distribution        Assignments        Resources       
Enzymes        Secretory Pathway        Apoptosis        OWW Basics

Enzyme Structure/Function: Background Information

The purification of an enzyme is often required in order to study its physical characteristics and the reaction it catalyzes. Purification is essentially a concentrating process in which the concentration of the target enzyme is increased while other proteins are eliminated. The enrichment process also removes metabolites and other cellular constituents that may influence the enzyme structure and/or the reaction. Background information on protein purification can be found below and also in your textbook.

During our first lab series, we will be partially purifying and characterizing the enzyme β-galactosidase from Escherichia coli, strain BL21(pET-14b). Although we will not be concerned with regulation of gene expression, it is of interest that experimentation on the production of β-galactosidase by E. coli ultimately led Francois Jacob and Jacques Monod to propose the 'operon' model for the regulation of gene expression. If you are interested in learning more about transcription regulation and early work on the genetic analysis of the lac-operon in E. coli, your textbook is a good resource.

To study protein structure and function, we have chosen the enzyme β-galactosidase for a variety of reasons. First, it is a convenient enzyme for learning basic techniques in enzymology because it is readily available in large quantities from microbial cells and is relatively stable. Next, the gene has been fully sequenced and cloned. The crystal structure of the protein and hence its 3-D configuration has been solved. Finally, the specific activity assay generally used to assess enzyme function is reliable and convenient (Jacobson et al., 1994; Ring, 1990; Wallenfels and Weil, 1972).

Βeta-galactosidase allows E. coli to grow in a culture medium containing the sugar lactose as the sole source of energy. The enzyme catalyzes the hydrolysis of lactose into glucose and galactose:


The purification of β-galactosidase is facilitated when E. coli produce large quantities of the enzyme. Growth on lactose as a carbon source is not a sufficient means of insuring abundant production of the enzyme since the expression of β-galactosidase is controlled. β-galactosidase production remains partially repressed even when cells are grown in the presence of the substrate lactose. It is possible, however, to transform genetically engineered strains of E. coli with a plasmid that expresses high levels of β-galactosidase. The plasmid fuses a strong promoter upstream of the β-galactosidase gene, lacZ. The gene promoter (and overexpression of the gene product) is regulated by isopropyl-β-D-thiogalactoside (IPTG), the structure of which is shown below.



In addition to allowing the production of large quantities of β-galactosidase, the lacZ gene on the plasmid, is modified so that the primary sequence of the enzyme is extended on the amino terminus with six histidines. This 6xHis tag can be exploited to purify the protein using metal chelate affinity chromatography (Kagedal, 1998), which we will perform in Lab 2.

Our goals for Lab 1 are:

  1. Calibrate our micropipets
  2. Induce E. coli BL21(pET-14b) cells to produce large quantities of 6xHis tagged β-galactosidase
  3. Learn how to model proteins and analyze their sequences, using the computer programs RasMol and Clustal Omega.



References:

Jacobson, R.H., Zhang, X.J., DuBose, R.F., and Matthews, B.W. (1994). Three-dimensional structure of β-galactosidase from E. coli. Nature 369, 761-6.

Karp, G. (2005). Cell and Molecular Biology Concepts and Experiments, 4th edition (Hoboken: John Wiley & Sons, Inc.).

Ring, M., Huber, R.E. (1990). Multiple replacements establish the importance of tyrosine-503 in beta-galactosidase (Escherichia coli). Arch Biochem Biophys 283, 342-50.

Wallenfels, K. and Weil, R. (1972). β-galactosidase. In The Enzymes, Vol VII, 3rd ed PD Boyer, ed. (New York: Academic Press), pp. 617-663.

Tutorials

RasMol Tutorial
Clustal Omega Tutorial

Protocols

THE INDUCTION OF β-GALACTOSIDASE

You will be provided with approximately 200 mL of E. coli BL21 (pET-14b) in log phase growth in Luria broth (LB) media. You will induce this genetically engineered strain to increase production of 6xHis-β-galactosidase by adding IPTG to the cells and incubating them at 37°C for one hour. You will collect the cells by centrifugation and freeze the cell pellet until the next lab period.

  1. Remove a flask containing the E. coli BL21(pET-14) cells from the 37°C shaking incubator, and bring it to your lab bench.
  2. Using a sterile 1 mL pipette and aseptic technique, remove 1.0 mL of the culture and add it to a microfuge tube labeled "pre-IPTG cells" on tape of your team color. Please also include the initials of your group and lab day. Centrifuge this small aliquot of cells for 2 minutes in a microcentrifuge. Discard the supernatant into the waste container next to the centrifuge and give the labeled dry pellet to your instructor to freeze at -80°C.
  3. Using a sterile 5.0 mL pipette and aseptic technique, add 2.0 mL of 0.1M IPTG to the flask of growing E. coli cells. This addition will yield a final concentration of approximately 1.0 mM IPTG in the culture flask.
  4. Return the flask of E. coli cells containing IPTG to the incubator and allow the cells to incubate with shaking for one hour. During this time IPTG will induce production of large amounts of β-galactosidase.
  5. Calibrate your micropipettes while your protein is synthesized by our genetically engineered strain of E. coli. Your instructor will demonstrate how to use the micropipets. Then follow the calibration protocol below.
  6. After the incubation period, remove the flask of E. coli from the incubator. Remove 1.0 mL of the culture to a microcentrifuge tube labeled "post-IPTG cells". Make sure the label includes the initials of your lab group, team color, & lab day. Pellet the cells as in step 2 above. Give the labeled tube containing the dry pellet to your instructor for freezing at -80°C.
  7. Pour the post-induction cells into an empty centrifuge bottle with a screw-cap provided by your lab instructor; secure the cap. Do not overfill the centrifuge bottle! If you have extra culture, it should be left in the flask to be autoclaved before disposal. Please do not throw any bacterial cells down the drain!
  8. Centrifuge the cells for 15 minutes at 8,000 RPM (~10,400x g) in a big Sorval centrifuge using the GSA rotor. Be sure that your bottle is balanced with another group’s before starting the centrifuge. Use sterile LB media for equalizing the weights.
  9. Carefully pour the supernatant back into the flask. Remove with a Pasteur pipette any media that remains. Label the bottle containing your cell pellet with the name of the cells (E. coli BL21(pET-14) + IPTG) on a piece of tape in your team color and include your initials and lab day; give your instructor the bottle to freeze at -80°C until the next lab period.

For a Word™ format protocol: Induction of β-gal


Protocol for Micropipette Calibration

  1. To calibrate your P 1000, P 200, and P 20 micropipettes, label 6 microcentrifuge tubes (1-6) and weigh them. Record the weights in the table below.
  2. Following the table below, pipet the specified volumes into the pre-weighed microcentrifuge tubes prepared above and then re-weigh them. Record all weights.
  3. Calculate the weight of the water in grams. 1000 μL of water should weigh 1 g at room temperature.
  4. If the water in any tube weighs more or less than 1 gram, ask your instructor for help. If your calibration is significantly off after several repeated attempts, your pipette (or your technique!) may need adjustment.


Tube # Tube Pre-Weight Vol. in μL using P20 Vol. in μL using P200 Vol. in μL using P1000 Weight of Tube + Water in grams Weight of Water in grams
1
10
0
990
2
0
100
900
3
20
175
805
4
2
88
910
5
0
200 (5 times)
0
6
20 (5 times)
0
900

For a Word™ format protocol: Micropipette calibration