Team:Caltech/Project/Lactose intolerance

From 2008.igem.org

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==Introduction==
==Introduction==
-
The gut flora of our digestive tract contains microorganisms that perform various useful functions for their hosts. Examples of such functions include growth inhibition of harmful microorganisms1, defense against the causes of many forms of Inflammatory Bowel Disease2, and the fermentation of carbohydrates and other molecules the human body cannot normally digest. Some bacterial strains, including the E. coli strain ‘Nissle 1917’, can persist in the gut of mice for months. Engineering these bacteria provide a new platform to treat various human diseases.  
+
The gut flora of our digestive tract contains microorganisms that perform various useful functions for their hosts. Examples of such functions include growth inhibition of harmful microorganisms<sup>1</sup>, defense against the causes of many forms of Inflammatory Bowel Disease<sup>2</sup>, and the fermentation of carbohydrates and other molecules the human body cannot normally digest. Some bacterial strains, including the ''Escherichia coli'' strain ‘Nissle 1917’, can persist in the gut of mice for months<sup>3</sup>. Engineering these bacteria provide a new platform to treat various human diseases.
Lactose intolerance is characterized by the inability to break down lactose in the small intestine. The undigested lactose instead passes to the large intestine, leading to two negative processes: osmotic imbalance and bacterial fermentation. High lactose levels raise the osmolarity of the colon, causing diarrhea. In addition, gut microbes metabolize the lactose into methane gas, causing abdominal pain. Both problems must be addressed in order to fully treat lactose intolerance. If we simply have our strain metabolize lactose, another strain will further ferment the byproducts, resulting in the same side effects.  
Lactose intolerance is characterized by the inability to break down lactose in the small intestine. The undigested lactose instead passes to the large intestine, leading to two negative processes: osmotic imbalance and bacterial fermentation. High lactose levels raise the osmolarity of the colon, causing diarrhea. In addition, gut microbes metabolize the lactose into methane gas, causing abdominal pain. Both problems must be addressed in order to fully treat lactose intolerance. If we simply have our strain metabolize lactose, another strain will further ferment the byproducts, resulting in the same side effects.  
-
Instead, we have our engineered strain allow the host to uptake lactose, clearing the sugar from the colon. To do this, we have engineered the ‘Nissle 1917’ strain to release ß-galactosidase, an enzyme that cleaves lactose into glucose and galactose. Since protein secretion is difficult in E. coli, our cells were engineered to lyse in order to release ß-galactosidase. However, lysis must occur only when lactose is present in the gut. Therefore, the cells also express a mutant lactose permease, allowing the strain to sense lactose in all conditions.  
+
Instead, we have our engineered strain allow the host to uptake lactose, clearing the sugar from the colon. To do this, we have engineered the ‘Nissle 1917’ strain to release ß-galactosidase, an enzyme that cleaves lactose into glucose and galactose. Since protein secretion is difficult in ''E. coli'', our cells were engineered to lyse in order to release ß-galactosidase. However, lysis must occur only when lactose is present in the gut. Therefore, the cells also express a mutant lactose permease, allowing the strain to sense lactose in all conditions.  
==System Design==
==System Design==
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[[image:Lactose_regulator.JPG‎|thumb|200px|left|A mutated LacY allows the uptake of lactose while LacZ produces large amounts of ß-galactosidase. The plasmid contains a strong constitutive promoter regulating both genes and a ColE1 replication origin.]]
[[image:Lactose_regulator.JPG‎|thumb|200px|left|A mutated LacY allows the uptake of lactose while LacZ produces large amounts of ß-galactosidase. The plasmid contains a strong constitutive promoter regulating both genes and a ColE1 replication origin.]]
The first plasmid consists of a synthetic lactose operon under strong constitutive expression. Our synthetic lactose operon encodes the ß-galactosidase LacZ and a mutant form of the lactose permease LacY. LacY is a membrane protein that actively transports lactose into the cell. Since most membrane proteins are toxic when overexpressed, we optimized our system to express appropriate levels of LacY without killing the cell. In the end, we want to express as much ß-galactosidase as we can to cleave as much lactose present in the large intestine.  
The first plasmid consists of a synthetic lactose operon under strong constitutive expression. Our synthetic lactose operon encodes the ß-galactosidase LacZ and a mutant form of the lactose permease LacY. LacY is a membrane protein that actively transports lactose into the cell. Since most membrane proteins are toxic when overexpressed, we optimized our system to express appropriate levels of LacY without killing the cell. In the end, we want to express as much ß-galactosidase as we can to cleave as much lactose present in the large intestine.  
-
The human gut is an unpredictable environment, and we wish our engineered cells to behave reliably despite this variability. E. coli are unable to uptake lactose in the presence of glucose, a phenomenon known as carbon catabolite repression. Catabolite repression is mediated by Enzyme IIA Glucose (IIAGluc), which inhibits the uptake of lactose in the presence of glucose by binding to LacY3. Previous research has identified various LacY mutations that prevent this inhibition and achieve increased uptake of lactose in the presence of glucose4.
+
The human gut is an unpredictable environment, and we wish our engineered cells to behave reliably despite this variability. E. coli are unable to uptake lactose in the presence of glucose, a phenomenon known as carbon catabolite repression. Catabolite repression is mediated by Enzyme IIA Glucose (IIAGluc), which inhibits the uptake of lactose in the presence of glucose by binding to LacY<sup>4</sup>. Previous research has identified various LacY mutations that prevent this inhibition and achieve increased uptake of lactose in the presence of glucose<sup>5</sup>.
-
<br><Br><Br><br><br><br><br><br>
+
{{clear}}
===Lactose-Induced Lysis===
===Lactose-Induced Lysis===
[[image:Lactose-inducible_lysis.JPG|thumb|200px|right|The lysis cassette plasmid acts as a lactose sensor. Intracellular lactose accumulation induces overexpression of the lysis cassette. Lactose inhibits binding of the LacI repressor to the promoter and P1 high copy origin of replication. A mutated holin gene in the lysis cassette allows faster lysis times.]]
[[image:Lactose-inducible_lysis.JPG|thumb|200px|right|The lysis cassette plasmid acts as a lactose sensor. Intracellular lactose accumulation induces overexpression of the lysis cassette. Lactose inhibits binding of the LacI repressor to the promoter and P1 high copy origin of replication. A mutated holin gene in the lysis cassette allows faster lysis times.]]
-
The second plasmid contains lactose inducible expression of λ phage lysis genes. To release ß-galactosidase, the cells will lyse when enough lactose is present in the cell to induce the expression of the lysis genes. Absent any stimulus, the plasmid containing the lysis genes remains at low copy but switches to high copy when induced with lactose. In addition, the lysis genes are placed behind lactose-inducible promoters. It is important that the lactose-inducible promoter be tightly regulated, since leaky expression will cause spontaneous lysis6.
+
The second plasmid contains lactose inducible expression of λ phage lysis genes. To release ß-galactosidase, the cells will lyse when enough lactose is present in the cell to induce the expression of the lysis genes. Absent any stimulus, the plasmid containing the lysis genes remains at low copy but switches to high copy when induced with lactose. In addition, the lysis genes are placed behind lactose-inducible promoters. It is important that the lactose-inducible promoter be tightly regulated, since leaky expression will cause spontaneous lysis<sup>6</sup>.
-
Using wild type λ phage lysis genes, lysis occurs 40-45 minutes after induction by lactose. Decreasing the lag time will reduce the extent of lactose fermentation and therefore produce fewer deleterious effects. Previous research has uncovered mutations that shorten the lysis time to approximately 10-15 minutes5, and these mutations will be incorporated into our final construct.
+
Using wild type λ phage lysis genes, lysis occurs 40-45 minutes after induction by lactose. Decreasing the lag time will reduce the extent of lactose fermentation and therefore produce fewer deleterious effects. Previous research has uncovered mutations that shorten the lysis time to approximately 10-15 minutes<sup>7</sup>, and these mutations will be incorporated into our final construct.
-
<br><Br><Br><br><br><br><br><br><BR><BR><BR>
+
{{clear}}
==Results==
==Results==
===Lactose Regulator===
===Lactose Regulator===
[[IMAGE:Promoter_strength.JPG|thumn|200px|left|Varying the expression levels of LacY with different promoters and ribosome binding sites. Cells died at our two highest expression levels, but survived on the other four. The construct with the weakest RBS was selected since we could express LacZ in the same operon with a strong promoter. This combination allows our cell to express high levels of LacZ and non-toxic levels of LacY.]]
[[IMAGE:Promoter_strength.JPG|thumn|200px|left|Varying the expression levels of LacY with different promoters and ribosome binding sites. Cells died at our two highest expression levels, but survived on the other four. The construct with the weakest RBS was selected since we could express LacZ in the same operon with a strong promoter. This combination allows our cell to express high levels of LacZ and non-toxic levels of LacY.]]
-
In the first plasmid, it was discovered that LacY is toxic when overexpressed. To determine safe expression levels, constructs were built with varying levels of LacY expression. Six plasmids were built from combinations of three different promoters and two different ribosome binding sites. The cells died when expressing the strongest and medium strength promoters along with the strongest ribosome binding site. We decided to express LacZ by combining the weakest ribosome binding site with the strongest constitutive promoter, preventing LacY toxicity while expressing LacZ in large quantities. <br>As mentioned earlier, our cells had to overcome carbon catabolite repression. Various mutations, including the insertion of two histidyl residues between amino acids S194 and A195 in the LacY gene, prevent the inhibition by IIAGluc. These insertions have been made, but we have not had enough time to test it. <br><BR><BR>
+
In the first plasmid, it was discovered that LacY is toxic when overexpressed. To determine safe expression levels, constructs were built with varying levels of LacY expression. Six plasmids were built from combinations of three different promoters and two different ribosome binding sites. The cells died when expressing the strongest and medium strength promoters along with the strongest ribosome binding site. We decided to express LacZ by combining the weakest ribosome binding site with the strongest constitutive promoter, preventing LacY toxicity while expressing LacZ in large quantities.  
 +
As mentioned earlier, our cells had to overcome carbon catabolite repression. Various mutations, including the insertion of two histidyl residues between amino acids S194 and A195 in the LacY gene, prevent the inhibition by IIAGluc. These insertions have been made, and tests are currently underway.
 +
{{clear}}
 +
 
 +
[[image:Figure_1_beta_assay.JPG‎|thumb|200px|right|ß-galactosidase activity of plasmid #1, with and without the strong constitutive promoter. Error bars on the data with a promoter show the standard deviation of two measurements.]]ß-galactosidase assays were used to quantify our data from our lactose regulator. The assay was performed twice on the synthetic network containing the lactose operon. The first assay was performed on a strain containing the plasmid lacking the strong constitutive promoter. This assay was performed to simulate the eventual system regulation. Our completed project will be under the expression of a promoter controlled by the FimE recombinase. The promoter will initially be pointing away from our synthetic lac operon and will be inverted to face the operon when the system is turned on. We saw significant levels of ß-galactosidase, a surprising result since the plasmid lacked a promoter. These levels are likely due to spurious transcription amplified by our strong ribosome binding site and high copy plasmid. We then repeated the assay one more time with the strong constitutive promoter to observe the dynamic range of LacZ expression in this system.
 +
{{clear}}
-
[[image:Figure_1_beta_assay.JPG‎|thumb|200px|right|ß-galactosidase activity of plasmid #1, with and without the strong constitutive promoter. Error bars on the data with a promoter show the standard deviation of two measurements.]]ß-galactosidase assays were used to quantify our data from our lactose regulator. The assay was performed twice on the synthetic network containing the lactose operon. The first assay was performed on a strain containing the plasmid lacking the strong constitutive promoter. This assay was performed to simulate the eventual system regulation. Our completed project will be under the expression of a promoter controlled by the FimE recombinase. The promoter will initially be pointing away from our synthetic lac operon and will be inverted to face the operon when the system is turned on. We saw significant levels of ß-galactosidase, a surprising result since the plasmid lacked a promoter. These levels are likely due to spurious transcription amplified by our strong ribosome binding site and high copy plasmid. We then repeated the assay one more time with the strong constitutive promoter to observe the dynamic range of LacZ expression in this system.<BR>
 
===Lactose-Induced Lysis===
===Lactose-Induced Lysis===
-
Our second plasmid was not completely cloned; therefore we were unable to perform assays on our second plasmid. There remains one more cloning step to place the lysis cassette into the vector containing the promoters. The single base mutations in the lysis cassette were not made, and if time allowed, they would have been completed as the final step.<br>
+
Our second plasmid was not completely cloned; therefore we were unable to perform assays on our second plasmid. There remains one more cloning step to place the lysis cassette into the vector containing the promoters. The single base mutations in the lysis cassette were not made, and if time allowed, they would have been completed as the final step.
==Discussion==
==Discussion==

Revision as of 16:20, 20 October 2008


iGEM 2008



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Curing Lactose Intolerance

Contents

Introduction

The gut flora of our digestive tract contains microorganisms that perform various useful functions for their hosts. Examples of such functions include growth inhibition of harmful microorganisms1, defense against the causes of many forms of Inflammatory Bowel Disease2, and the fermentation of carbohydrates and other molecules the human body cannot normally digest. Some bacterial strains, including the Escherichia coli strain ‘Nissle 1917’, can persist in the gut of mice for months3. Engineering these bacteria provide a new platform to treat various human diseases. Lactose intolerance is characterized by the inability to break down lactose in the small intestine. The undigested lactose instead passes to the large intestine, leading to two negative processes: osmotic imbalance and bacterial fermentation. High lactose levels raise the osmolarity of the colon, causing diarrhea. In addition, gut microbes metabolize the lactose into methane gas, causing abdominal pain. Both problems must be addressed in order to fully treat lactose intolerance. If we simply have our strain metabolize lactose, another strain will further ferment the byproducts, resulting in the same side effects. Instead, we have our engineered strain allow the host to uptake lactose, clearing the sugar from the colon. To do this, we have engineered the ‘Nissle 1917’ strain to release ß-galactosidase, an enzyme that cleaves lactose into glucose and galactose. Since protein secretion is difficult in E. coli, our cells were engineered to lyse in order to release ß-galactosidase. However, lysis must occur only when lactose is present in the gut. Therefore, the cells also express a mutant lactose permease, allowing the strain to sense lactose in all conditions.

System Design

Lactose Regulator

A mutated LacY allows the uptake of lactose while LacZ produces large amounts of ß-galactosidase. The plasmid contains a strong constitutive promoter regulating both genes and a ColE1 replication origin.

The first plasmid consists of a synthetic lactose operon under strong constitutive expression. Our synthetic lactose operon encodes the ß-galactosidase LacZ and a mutant form of the lactose permease LacY. LacY is a membrane protein that actively transports lactose into the cell. Since most membrane proteins are toxic when overexpressed, we optimized our system to express appropriate levels of LacY without killing the cell. In the end, we want to express as much ß-galactosidase as we can to cleave as much lactose present in the large intestine. The human gut is an unpredictable environment, and we wish our engineered cells to behave reliably despite this variability. E. coli are unable to uptake lactose in the presence of glucose, a phenomenon known as carbon catabolite repression. Catabolite repression is mediated by Enzyme IIA Glucose (IIAGluc), which inhibits the uptake of lactose in the presence of glucose by binding to LacY4. Previous research has identified various LacY mutations that prevent this inhibition and achieve increased uptake of lactose in the presence of glucose5.

Lactose-Induced Lysis

The lysis cassette plasmid acts as a lactose sensor. Intracellular lactose accumulation induces overexpression of the lysis cassette. Lactose inhibits binding of the LacI repressor to the promoter and P1 high copy origin of replication. A mutated holin gene in the lysis cassette allows faster lysis times.

The second plasmid contains lactose inducible expression of λ phage lysis genes. To release ß-galactosidase, the cells will lyse when enough lactose is present in the cell to induce the expression of the lysis genes. Absent any stimulus, the plasmid containing the lysis genes remains at low copy but switches to high copy when induced with lactose. In addition, the lysis genes are placed behind lactose-inducible promoters. It is important that the lactose-inducible promoter be tightly regulated, since leaky expression will cause spontaneous lysis6. Using wild type λ phage lysis genes, lysis occurs 40-45 minutes after induction by lactose. Decreasing the lag time will reduce the extent of lactose fermentation and therefore produce fewer deleterious effects. Previous research has uncovered mutations that shorten the lysis time to approximately 10-15 minutes7, and these mutations will be incorporated into our final construct.

Results

Lactose Regulator

Varying the expression levels of LacY with different promoters and ribosome binding sites. Cells died at our two highest expression levels, but survived on the other four. The construct with the weakest RBS was selected since we could express LacZ in the same operon with a strong promoter. This combination allows our cell to express high levels of LacZ and non-toxic levels of LacY.

In the first plasmid, it was discovered that LacY is toxic when overexpressed. To determine safe expression levels, constructs were built with varying levels of LacY expression. Six plasmids were built from combinations of three different promoters and two different ribosome binding sites. The cells died when expressing the strongest and medium strength promoters along with the strongest ribosome binding site. We decided to express LacZ by combining the weakest ribosome binding site with the strongest constitutive promoter, preventing LacY toxicity while expressing LacZ in large quantities. As mentioned earlier, our cells had to overcome carbon catabolite repression. Various mutations, including the insertion of two histidyl residues between amino acids S194 and A195 in the LacY gene, prevent the inhibition by IIAGluc. These insertions have been made, and tests are currently underway.

ß-galactosidase activity of plasmid #1, with and without the strong constitutive promoter. Error bars on the data with a promoter show the standard deviation of two measurements.
ß-galactosidase assays were used to quantify our data from our lactose regulator. The assay was performed twice on the synthetic network containing the lactose operon. The first assay was performed on a strain containing the plasmid lacking the strong constitutive promoter. This assay was performed to simulate the eventual system regulation. Our completed project will be under the expression of a promoter controlled by the FimE recombinase. The promoter will initially be pointing away from our synthetic lac operon and will be inverted to face the operon when the system is turned on. We saw significant levels of ß-galactosidase, a surprising result since the plasmid lacked a promoter. These levels are likely due to spurious transcription amplified by our strong ribosome binding site and high copy plasmid. We then repeated the assay one more time with the strong constitutive promoter to observe the dynamic range of LacZ expression in this system.

Lactose-Induced Lysis

Our second plasmid was not completely cloned; therefore we were unable to perform assays on our second plasmid. There remains one more cloning step to place the lysis cassette into the vector containing the promoters. The single base mutations in the lysis cassette were not made, and if time allowed, they would have been completed as the final step.

Discussion

Our final system was not completely finished; however, we have made progress. The lactose regulator plasmid was completely constructed with the insertion of two histidyl residues in their appropriate locations. Our lactose-induced lysis plasmid was one cloning step away from being completed. The promoter and flanking terminator were cloned in parallel with the lysis cassette and a terminator. The last cloning step would be to ligate the two together. In addition, the two separate point mutations have not been made, and the final step in our cloning would be to add those mutations.
After completing the construction phase, we would characterize our system, and then move our complete network into the ‘Nissle 1917’ strain. A construct containing our lactose inducible promoters and GFP would show our promoters can maintain tight expression of GFP from uninduced to induced. We would combine this construct with our first plasmid containing the mutated LacY, to show we can achieve induction by lactose even in the presence of glucose. To show the effect our second plasmid, it would be necessary to achieve lysis with lactose. In addition, we want to show that we can achieve lysis with lactose in the presence of glucose. Finally, the final assay we want to perform on our system would show our cells able to uptake lactose in the presence of glucose, and lyse soon after. Once the cells lyse, the cell lysis should contain ß-galactosidase at levels close to what we received from our ß-galactosidase assay on our lactose regulator.

Methods

Methods can be found here

References

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