"
Team:WHU-China/Project/fatty acid degradation
From 2012.igem.org
| Line 1: | Line 1: | ||
<HTML> | <HTML> | ||
| - | < | + | <h1> |
Fatty Acid Degradation Device | Fatty Acid Degradation Device | ||
| - | </ | + | </h1> |
| - | < | + | <h2> |
Purpose | Purpose | ||
| - | </ | + | </h2> |
<p> | <p> | ||
To help people lose weight without the need of food restriction. We designed a genetically modified <i>E.coli</i> that can sense and degrade excessive fatty acid intake by the host. We hope that, together with other two devices we designed, we can introduce our <i>E.coslim</i> as resident in intestine to consume the excessive calories intake by the host and regulate intestinal microbiota. | To help people lose weight without the need of food restriction. We designed a genetically modified <i>E.coli</i> that can sense and degrade excessive fatty acid intake by the host. We hope that, together with other two devices we designed, we can introduce our <i>E.coslim</i> as resident in intestine to consume the excessive calories intake by the host and regulate intestinal microbiota. | ||
</p> | </p> | ||
| - | < | + | <h2> |
Outline | Outline | ||
| - | </ | + | </h2> |
| - | <P>Genes that are responsible for degradation and transportation of fatty acids (FAs) from <i>E.coli K12</i> and from <i>Salmonella enterica LT2</i> were cloned. Also, promoter that can under the sole regulation of fatty acids was also designed. By placing those fatty acids degradation genes downstream of the artificially designed promoter that can sense the concentration of FAs. We hope to create a device that to degrade FAs only when the concentration of FAs is high. | + | <P> |
| + | <img src="https://static.igem.org/mediawiki/2012/7/76/Fatty_Acid_M.png" width="300" height="400" hspace="2" vspace="1" border="2" style="float:left" /> | ||
| + | |||
| + | Genes that are responsible for degradation and transportation of fatty acids (FAs) from <i>E.coli K12</i> and from <i>Salmonella enterica LT2</i> were cloned. Also, promoter that can under the sole regulation of fatty acids was also designed. By placing those fatty acids degradation genes downstream of the artificially designed promoter that can sense the concentration of FAs. We hope to create a device that to degrade FAs only when the concentration of FAs is high. | ||
</p> | </p> | ||
<p> | <p> | ||
| - | |||
Long chain fatty acids are firstly being imported by the transmembrane protein FadL. After FAs get into cells, a CoA will be added by inner membrane-associated FadD (acyl-CoA synthase). β-oxidation is initiated by FadE(acyl-CoA dehydrogenase), which will convert acyl-CoA into enoyl-CoA. The followed cycles of hydration, oxidation, and thiolytic cleavage are carried out by tetrameric complex consisting of two FadA and two FadB proteins or two FadI and two FadJ in anaerobic condition. FadR is a transcriptional regulator that, when not binds to acyl-CoA, can either serve as activator for fatty acid synthetase gene like FabA, FabB and etc. or repressor for fatty acid degradation gene like FadA, FadB, FadD FadE, FadL, FadI, FadJ and etc. After long chain fatty acid is converted to fatty acyl- CoA by FadD, it can bind to FadR. The binding will alter the conformation of FadR, making FadR unable to bind to the DNA sequence it recognizes to fulfill its function. Therefore, FadR can no longer activate or repress the transcription of genes downstream FadR binding sites. However, to our knowledge, there is no promoter exists in nature that can respond solely to FadR since those promoters are often regulated by glucose concentration or oxidative stress and many other factors.</br> | Long chain fatty acids are firstly being imported by the transmembrane protein FadL. After FAs get into cells, a CoA will be added by inner membrane-associated FadD (acyl-CoA synthase). β-oxidation is initiated by FadE(acyl-CoA dehydrogenase), which will convert acyl-CoA into enoyl-CoA. The followed cycles of hydration, oxidation, and thiolytic cleavage are carried out by tetrameric complex consisting of two FadA and two FadB proteins or two FadI and two FadJ in anaerobic condition. FadR is a transcriptional regulator that, when not binds to acyl-CoA, can either serve as activator for fatty acid synthetase gene like FabA, FabB and etc. or repressor for fatty acid degradation gene like FadA, FadB, FadD FadE, FadL, FadI, FadJ and etc. After long chain fatty acid is converted to fatty acyl- CoA by FadD, it can bind to FadR. The binding will alter the conformation of FadR, making FadR unable to bind to the DNA sequence it recognizes to fulfill its function. Therefore, FadR can no longer activate or repress the transcription of genes downstream FadR binding sites. However, to our knowledge, there is no promoter exists in nature that can respond solely to FadR since those promoters are often regulated by glucose concentration or oxidative stress and many other factors.</br> | ||
| - | In our design, FadL, FadD, FadE, FadA, FadB FadI, FadJ and FadA from <i>Escherichia coli K12</i>, and FadA, FadB and FadE from <i>Salmonella enterica LT2</i> are placed downstream a synthetic promoter PfadR (BBa_K861060) to make them under the sole regulation of fatty acids concentration. | + | In our design, FadL, FadD, FadE, FadA, FadB FadI, FadJ and FadA from <i>Escherichia coli K12</i>, and FadA, FadB and FadE from <i>Salmonella enterica LT2</i> are placed downstream a synthetic promoter PfadR (BBa_K861060) to make them under the sole regulation of fatty acids concentration.</br> |
| + | </br> | ||
| + | </p> | ||
| + | <h2> | ||
| + | Progress | ||
| + | </h2> | ||
| + | <h3> | ||
| + | Cloning of the gene | ||
| + | </h3> | ||
| + | <p> | ||
| + | First, the genome of <i>Escherichia coli K12 str. DH5ɑ</i> and <i>Salmonella enterica LT2</i> (symbolized as S-) were got and amplified in PCR using primer for each gene. The sequences of the primers used are as bellow (5’---3’).</br> | ||
| - | </ | + | FadR Forward: GGAATTCTCTAGAATGGTCATTAAGGCGCAAAG</br> |
| + | Reverse: GACTAGTCTTATCGCCCCTGAATGGCTAAATC</br> | ||
| + | FadA Forward: GGAATTCTCTAGAATGGAACAGGTTGTCATTGTCG</br> | ||
| + | Reverse: GACTAGTTTAAACCCGCTCAAACACCGT</br> | ||
| + | |||
| + | FadB Forward: GGAATTCTCTAGAATGCTTTACAAAGGCGACACC</br> | ||
| + | Reverse: GACTAGTTTAAGCCGTTTTCAGGTCGCC</br> | ||
| + | |||
| + | FadD Forward: GGAATTC TCTAGATTGAAGAAGGTTTGGCTTAACCG</br> | ||
| + | Reverse: GACTAGTTCAGGCTTTATTGTCCACTTTGC</br> | ||
| + | |||
| + | FadE Forward: GGAATTC TCTAGAATGATGATTTTGAGTATTCTCG</br> | ||
| + | Reverse: GACTAGTTTACGCGGCTTCAACTTTCCG</br> | ||
| + | |||
| + | FadL Forward: GGAATTC TCTAGAATGAGCCAGAAAACCCTG</br> | ||
| + | Reverse: GACTAGTTAGAACGCGTAGTTAAAGTTAG</br> | ||
| + | |||
| + | FadI Forward: GGAATTC TCTAGA ATGGGTCAGGTTTTACC</br> | ||
| + | Reverse: GACTAGTTTATTCCGCCTCCAGAACCA</br> | ||
| + | |||
| + | FadJ Forward: GGAATTCTCTAGAATGGAAATGACATCAGC</br> | ||
| + | Reverse: GACTAGTTTATTGCAGGTCAGTTGCAGTTG</br> | ||
| + | |||
| + | S-FadA Forward: GGAATTCTCTAGAATGGTCATTAAGGCGCAAAG</br> | ||
| + | Reverse: GACTAGTCTTATCGCCCCTGAATGGCTAAATC</br> | ||
| + | |||
| + | S-FadB Forward: GGAATTCTCTAGAATGCTTTATAAAGGCGACACC</br> | ||
| + | Reverse: GACTAGTTAAGCCGTTTTCAGAGAACC</br> | ||
| + | |||
| + | S-FadE Forward: GGAATTCTCTAGAATGATGATTTTGAGTATTATCG</br> | ||
| + | Reverse: GACTAGTTATGCGGCTTCGACTTTACGC</br> | ||
| + | |||
| + | </p> | ||
<h3> | <h3> | ||
| - | + | Design of the Promoter PfadR Repressed by Fatty Acids | |
</h3> | </h3> | ||
| + | <p> | ||
| + | Promoter PfadR, is derived from BBa_J23110. Specifically, FadR binding site of FadL gene is placed overlapped with the last 3 bases of BBa_J23110 The sequence was synthesized with restriction sites for EcoRI and XbaI at the 5' terminal and SpeI at 3' terminal. We use overlapping PCR to get the double strand DNA. The sequence design of PfadR is as followed:</br> | ||
| + | Forward: | ||
| + | GGAATTCTCTAGATTTACGGCTAGCTCAGTCCTAGGTACAATGCTAGCTGGTCCGACCT</br> | ||
| + | Reverse： | ||
| + | GACTAGTTCTTAGAAATCAGACCAGTGGCGAGAGTATAGGTCGGACCAGCTAGCATTGT | ||
| + | |||
| + | </p> | ||
</HTML> | </HTML> | ||
Revision as of 03:42, 14 September 2012
Fatty Acid Degradation Device
Purpose
To help people lose weight without the need of food restriction. We designed a genetically modified E.coli that can sense and degrade excessive fatty acid intake by the host. We hope that, together with other two devices we designed, we can introduce our E.coslim as resident in intestine to consume the excessive calories intake by the host and regulate intestinal microbiota.
Outline
Genes that are responsible for degradation and transportation of fatty acids (FAs) from E.coli K12 and from Salmonella enterica LT2 were cloned. Also, promoter that can under the sole regulation of fatty acids was also designed. By placing those fatty acids degradation genes downstream of the artificially designed promoter that can sense the concentration of FAs. We hope to create a device that to degrade FAs only when the concentration of FAs is high.
Long chain fatty acids are firstly being imported by the transmembrane protein FadL. After FAs get into cells, a CoA will be added by inner membrane-associated FadD (acyl-CoA synthase). β-oxidation is initiated by FadE(acyl-CoA dehydrogenase), which will convert acyl-CoA into enoyl-CoA. The followed cycles of hydration, oxidation, and thiolytic cleavage are carried out by tetrameric complex consisting of two FadA and two FadB proteins or two FadI and two FadJ in anaerobic condition. FadR is a transcriptional regulator that, when not binds to acyl-CoA, can either serve as activator for fatty acid synthetase gene like FabA, FabB and etc. or repressor for fatty acid degradation gene like FadA, FadB, FadD FadE, FadL, FadI, FadJ and etc. After long chain fatty acid is converted to fatty acyl- CoA by FadD, it can bind to FadR. The binding will alter the conformation of FadR, making FadR unable to bind to the DNA sequence it recognizes to fulfill its function. Therefore, FadR can no longer activate or repress the transcription of genes downstream FadR binding sites. However, to our knowledge, there is no promoter exists in nature that can respond solely to FadR since those promoters are often regulated by glucose concentration or oxidative stress and many other factors. In our design, FadL, FadD, FadE, FadA, FadB FadI, FadJ and FadA from Escherichia coli K12, and FadA, FadB and FadE from Salmonella enterica LT2 are placed downstream a synthetic promoter PfadR (BBa_K861060) to make them under the sole regulation of fatty acids concentration.
Progress
Cloning of the gene
First, the genome of Escherichia coli K12 str. DH5ɑ and Salmonella enterica LT2 (symbolized as S-) were got and amplified in PCR using primer for each gene. The sequences of the primers used are as bellow (5’---3’). FadR Forward: GGAATTCTCTAGAATGGTCATTAAGGCGCAAAG Reverse: GACTAGTCTTATCGCCCCTGAATGGCTAAATC FadA Forward: GGAATTCTCTAGAATGGAACAGGTTGTCATTGTCG Reverse: GACTAGTTTAAACCCGCTCAAACACCGT FadB Forward: GGAATTCTCTAGAATGCTTTACAAAGGCGACACC Reverse: GACTAGTTTAAGCCGTTTTCAGGTCGCC FadD Forward: GGAATTC TCTAGATTGAAGAAGGTTTGGCTTAACCG Reverse: GACTAGTTCAGGCTTTATTGTCCACTTTGC FadE Forward: GGAATTC TCTAGAATGATGATTTTGAGTATTCTCG Reverse: GACTAGTTTACGCGGCTTCAACTTTCCG FadL Forward: GGAATTC TCTAGAATGAGCCAGAAAACCCTG Reverse: GACTAGTTAGAACGCGTAGTTAAAGTTAG FadI Forward: GGAATTC TCTAGA ATGGGTCAGGTTTTACC Reverse: GACTAGTTTATTCCGCCTCCAGAACCA FadJ Forward: GGAATTCTCTAGAATGGAAATGACATCAGC Reverse: GACTAGTTTATTGCAGGTCAGTTGCAGTTG S-FadA Forward: GGAATTCTCTAGAATGGTCATTAAGGCGCAAAG Reverse: GACTAGTCTTATCGCCCCTGAATGGCTAAATC S-FadB Forward: GGAATTCTCTAGAATGCTTTATAAAGGCGACACC Reverse: GACTAGTTAAGCCGTTTTCAGAGAACC S-FadE Forward: GGAATTCTCTAGAATGATGATTTTGAGTATTATCG Reverse: GACTAGTTATGCGGCTTCGACTTTACGC
Design of the Promoter PfadR Repressed by Fatty Acids
Promoter PfadR, is derived from BBa_J23110. Specifically, FadR binding site of FadL gene is placed overlapped with the last 3 bases of BBa_J23110 The sequence was synthesized with restriction sites for EcoRI and XbaI at the 5' terminal and SpeI at 3' terminal. We use overlapping PCR to get the double strand DNA. The sequence design of PfadR is as followed: Forward: GGAATTCTCTAGATTTACGGCTAGCTCAGTCCTAGGTACAATGCTAGCTGGTCCGACCT Reverse： GACTAGTTCTTAGAAATCAGACCAGTGGCGAGAGTATAGGTCGGACCAGCTAGCATTGT
