IGEM:Caltech/2008/Project/Vitamins

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==Folate Biosynthesis Pathway==
==Folate Biosynthesis Pathway==
-
[[Image:folate_gene_cluster.jpg |frame|center|50px|The folate gene cluster from "L.lactis". Black arrows represent genes which have been tested in metabolic engineering studies, shaded arrows represent genes involved in folate biosynthesis, and white arrows represent genes not involved in folate synthesis. <cite>sybesma1</cite>]]
+
[[Image:folate_gene_cluster.jpg |frame|center|50px|The folate gene cluster from ''L.lactis''. Black arrows represent genes which have been tested in metabolic engineering studies, shaded arrows represent genes involved in folate biosynthesis, and white arrows represent genes not involved in folate synthesis. <cite>sybesma1</cite>]]
-
Although folate is naturally produced in "E.coli", the folate biosynthesis pathway in the bacteria "Lactococcus lactis" has been more heavily characterized and studied. There are six major enzymes involved in folate synthesis, which, in "L.lactis", are contained in five genes: "folB", "folKE", "folP", "folC", and "folA"<cite>sybesma1</cite>. The first four, which we have chosen to focus on, are located in a gene cluster approximately 4.4kb long. We’ve chosen not to focus on "folA" for the time being because "folA" encodes an enzyme which turns one form of folate (dihydrofolate) into another form of folate(tetrahydrofolate). Since our assay would detect both types of folate as part of the total folate production, "folA" was not a prime target for overexpression of folate. In previous studies, this folate gene cluster has been successfully transformed into the folate-consuming bacteria "L.gasseri", turning the bacteria in to folate-producers<cite>wegkamp1</cite>. Therefore, we have chosen to also use the folate operon from L.lactis, which also offers the additional benefit of removing the operon from its natural regulatory context.  
+
Although folate is naturally produced in ''E.coli'', the folate biosynthesis pathway in the bacteria ''Lactococcus lactis'' has been more heavily characterized and studied. There are six major enzymes involved in folate synthesis, which, in ''L.lactis'', are contained in five genes: ''folB'', ''folKE'', ''folP'', ''folC'', and ''folA''<cite>sybesma1</cite>. The first four, which we have chosen to focus on, are located in a gene cluster approximately 4.4kb long. We’ve chosen not to focus on ''folA'' for the time being because ''folA'' encodes an enzyme which turns one form of folate (dihydrofolate) into another form of folate(tetrahydrofolate). Since our assay would detect both types of folate as part of the total folate production, ''folA'' was not a prime target for overexpression of folate. In previous studies, this folate gene cluster has been successfully transformed into the folate-consuming bacteria ''L.gasseri'', turning the bacteria in to folate-producers<cite>wegkamp1</cite>. Therefore, we have chosen to also use the folate operon from L.lactis, which also offers the additional benefit of removing the operon from its natural regulatory context.  
[[Image:Llactis_folate_synthesis.jpg|thumb|left|The folate biosynthesis pathway from ''L.lactis''. <cite>sybesma1</cite>]]
[[Image:Llactis_folate_synthesis.jpg|thumb|left|The folate biosynthesis pathway from ''L.lactis''. <cite>sybesma1</cite>]]
-
Our strategy is to clone the entire folate operon from the "L.lactis" genome and to transform the entire operon into "E.coli". However, because we are unsure of whether or not the ribosomal binding sites (RBS) within the "L.lactis" operon would work in E.coli, we are also going to unpack the operon by cloning each of the four genes individually, placing them behind "E.coli" RBSs, and then recombine them into a single empty BioBricks™ plasmid. In addition to the main folate operon, we will also be experimenting with overexpression of the para-aminobenzoic acid (pABA) synthesis pathway from chorismate. Wegkamp "et al." have shown that in order to increase overall total levels of folate, both the pABA synthesis genes and certain folate production genes need to be overexpressed<cite>wegkamp2</cite>. The pABA pathway involves three genes, "pabA", "pabB", and "pabC" – though in "L.lactis", "pabB" is actually a fusion gene encoding for both "pabB" and "pabC"<cite>wegkamp2</cite>.
+
Our strategy is to clone the entire folate operon from the ''L.lactis'' genome and to transform the entire operon into ''E.coli''. However, because we are unsure of whether or not the ribosomal binding sites (RBS) within the ''L.lactis'' operon would work in ''E.coli'', we are also going to unpack the operon by cloning each of the four genes individually, placing them behind ''E.coli'' RBSs, and then recombine them into a single empty BioBricks™ plasmid. In addition to the main folate operon, we will also be experimenting with overexpression of the para-aminobenzoic acid (pABA) synthesis pathway from chorismate. Wegkamp ''et al.'' have shown that in order to increase overall total levels of folate, both the pABA synthesis genes and certain folate production genes need to be overexpressed<cite>wegkamp2</cite>. The pABA pathway involves three genes, ''pabA'', ''pabB'', and ''pabC'' – though in ''L.lactis'', ''pabB'' is actually a fusion gene encoding for both ''pabB'' and ''pabC''<cite>wegkamp2</cite>.
==System Design==
==System Design==
[[Image:systemdesign_folate.png|thumb|left|Final folate biosynthesis plasmid]][[Image:systemdesign_paba.png|thumb|left|Final folate biosynthesis plasmid]]
[[Image:systemdesign_folate.png|thumb|left|Final folate biosynthesis plasmid]][[Image:systemdesign_paba.png|thumb|left|Final folate biosynthesis plasmid]]
-
The overall system design for testing folate production in "E.coli" is to construct two plasmids – one for the folate biosynthesis pathway, and one for the pABA synthesis pathway. In addition to ensuring that the plasmids are complementary, each plasmid would need to contain a different variable copy origin of replication, which would be low copy by default, but can be switched to high copy via the addition of Isopropyl β-D-1-thiogalactopyranoside (IPTG)to the media. This will allow us to test overexpression of each plasmid separately. In addition, each plasmid will contain a constitutive promoter, since we would want folate to be produced constantly. The purple dots represent ribosomal binding sites (RBS), followed by the gene (green arrow), and eventually terminating in a double stop (TAATAA) sequence, as regulated by the Registry of Standard Biological Parts.
+
The overall system design for testing folate production in ''E.coli'' is to construct two plasmids – one for the folate biosynthesis pathway, and one for the pABA synthesis pathway. In addition to ensuring that the plasmids are complementary, each plasmid would need to contain a different variable copy origin of replication, which would be low copy by default, but can be switched to high copy via the addition of Isopropyl β-D-1-thiogalactopyranoside (IPTG)to the media. This will allow us to test overexpression of each plasmid separately. In addition, each plasmid will contain a constitutive promoter, since we would want folate to be produced constantly. The purple dots represent ribosomal binding sites (RBS), followed by the gene (green arrow), and eventually terminating in a double stop (TAATAA) sequence, as regulated by the Registry of Standard Biological Parts.
==Folate Detection Methods==
==Folate Detection Methods==
-
We will be detecting folate production, and thus the relative success of our engineering efforts, via a microbiological assay involving the folate-dependent strain "Enterococcus hirae"<cite>horne</cite>. This assay involves first the characterization of a standard growth curve of "E. hirae" given known quantities of folate present in the growth media. Once the standard curve has been established, then experimental growth levels, as quantified by spectrophotometry, can be interpolated. PABA concentrations will be measured via high performance liquid chromatography (HPLC) <cite>wegkamp2</cite>.
+
We will be detecting folate production, and thus the relative success of our engineering efforts, via a microbiological assay involving the folate-dependent strain ''Enterococcus hirae''<cite>horne</cite>. This assay involves first the characterization of a standard growth curve of ''E. hirae'' given known quantities of folate present in the growth media. Once the standard curve has been established, then experimental growth levels, as quantified by spectrophotometry, can be interpolated. PABA concentrations will be measured via high performance liquid chromatography (HPLC) <cite>wegkamp2</cite>.
 +
 
 +
[[Folate microbiological assay protocol]]
 +
 
 +
[[para-aminobenzoic acid (pABA) HPLC protocol]]
-
Folate microbiological assay protocol --> future link here when i figure out how to make a new page.
 
==Relevant Registry Parts==
==Relevant Registry Parts==
 +
===Basic Parts===
{| border="1"
{| border="1"
|+ Basic Parts (Extracted from ''L.lactis subspe. IL1403'' genome)
|+ Basic Parts (Extracted from ''L.lactis subspe. IL1403'' genome)
Line 63: Line 67:
|-
|-
|}
|}
-
 
+
===Construction Intermediates===
{| border="1"
{| border="1"
|+ Construction Intermediates: Adding b0034 (strong RBS) before each individual gene
|+ Construction Intermediates: Adding b0034 (strong RBS) before each individual gene
Line 89: Line 93:
| [http://partsregistry.org/Part:BBa_S03961 BBa_S03961]|| Combining folB (with RBS) + folKE (with RBS) || YES|| YES
| [http://partsregistry.org/Part:BBa_S03961 BBa_S03961]|| Combining folB (with RBS) + folKE (with RBS) || YES|| YES
|-
|-
 +
|}
 +
{| border="1"
 +
|+ Construction Intermediates: Adding j23100 (constitutive promoter) to each gene (with RBS already)
 +
! Part Name !! Registry # !! Description !! Cloned? !! Sequence confirmed? 
 +
|-
 +
! folB + j23100
 +
| [http://partsregistry.org/Part:BBa_S04032 BBa_S04032]|| Promoter(j23100) + RBS(b0034) + dihydroneopterin aldolase || YES|| YES
 +
|-
 +
! folKE + j23100
 +
| [http://partsregistry.org/Part:BBa_S04033 BBa_S04033]|| Promoter + RBS + fusion gene: GTP cyclohydrolase & 2-amino-4-hydroxy-6- hydroxymethyldihydropteridine pyrophosphokinase  || YES|| YES
 +
|-
 +
! pabA + j23100
 +
| [http://partsregistry.org/Part:BBa_S04034 BBa_S04034]|| Promoter + RBS + para-aminobenzoate synthetase component II || YES|| YES
 +
|-
 +
! folBKE + j23100
 +
| [http://partsregistry.org/Part:BBa_S04035 BBa_S04035]|| Promoter + folB (with RBS) + folKE (with RBS) || YES || YES
 +
|-
 +
! pabB + j23100
 +
| [http://partsregistry.org/Part:BBa_S04039 BBa_S04039]|| Promoter + pabB  || YES || YES
 +
|-
 +
! pabB + b0015
 +
| [http://partsregistry.org/Part:BBa_S04038 BBa_S04038]|| pabB + b0015 terminator || MAYBE || WAITING
 +
|-
 +
|}
 +
===Composite Parts===
 +
{| border="1"
 +
|+ Composite Parts: Adding b0015 (double terminator) to complete constructs with promoter + RBS already
 +
! Part Name !! Registry # !! Description !! Cloned? !! Sequence confirmed? 
 +
|-
 +
! folB + b0015
 +
| [http://partsregistry.org/Part:BBa_K137053 BBa_K137053]|| Promoter(j23100) + RBS(b0034) + folB (dihydroneopterin aldolase) + double terminator (b0015) || YES || YES
 +
|-
 +
! folKE + b0015
 +
| [http://partsregistry.org/Part:BBa_K137054 BBa_K137054]|| Promoter + RBS + folKE (fusion gene: GTP cyclohydrolase & 2-amino-4-hydroxy-6- hydroxymethyldihydropteridine pyrophosphokinase) + double terminator (b0015)  || NO || NO
 +
|-
 +
! pabA + b0015
 +
| [http://partsregistry.org/Part:BBa_K137055 BBa_K137055]|| Promoter + RBS + pabA (para-aminobenzoate synthetase component II) || Maybe  || waiting
 +
|-
 +
! folBKE + b0015
 +
| [http://partsregistry.org/Part:BBa_K137056 BBa_K137056]|| Promoter + RBS + folB + folKE + b0015 || NO || NO
 +
|-
|}
|}
==Current Progress==
==Current Progress==
-
*7/10/08  
+
*8/01/08  
 +
**On the last cloning step (adding B0015 to everything). Once that's finished I can actually start the metabolic engineering part of the project...
 +
 
 +
* [[Media:Vhsiao_Abstract_Final_V3.pdf |Abstract for SFP Summer Session]]
 +
* [[Media:Amgen_Progress_Report_1.pdf | Progress Report #1]]
 +
* [[Media:Final_Amgen_Progress_Report_2.pdf | Progress Report #2]]
==References==
==References==

Current revision


iGEM 2008

Home        People        Project        Protocols        Notes        Ideas        Changes       


Contents

Vitamin production

Background Information on Folate

The structure of tetrahydrofolate [1]
The structure of tetrahydrofolate [1]

Folate, the generic term for the various forms of Vitamin B9, is an essential vitamin because it is heavily involved in amino acid synthesis as well as single-carbon transfer reactions. Folate deficiencies in women can result in birth defects such as neural tube defects and other spinal cord malformations. As important as folate is, humans are unable to produce folate, and so must obtain it from eating foods such as green leafy vegetables or folate-fortified cereals [1]. An engineered strain of bacteria that would constantly release folate into the gut would reduce the need to fortify breads and cereals with folate, as well as reduce folate-related birth defects in regions with little access to folate-containing foods. In addition to all the reasons stated above, folate is an ideal vitamin to be produced in the gut because, unlike many other vitamins, it has been shown to be absorbed in physiologically relevant quantities in the large intestine[2].

Structurally, folate consists of three main parts: pteridine, p-aminobenzoic acid (pABA), and L-glutamate.


Folate Biosynthesis Pathway

The folate gene cluster from L.lactis. Black arrows represent genes which have been tested in metabolic engineering studies, shaded arrows represent genes involved in folate biosynthesis, and white arrows represent genes not involved in folate synthesis. [1]
The folate gene cluster from L.lactis. Black arrows represent genes which have been tested in metabolic engineering studies, shaded arrows represent genes involved in folate biosynthesis, and white arrows represent genes not involved in folate synthesis. [1]

Although folate is naturally produced in E.coli, the folate biosynthesis pathway in the bacteria Lactococcus lactis has been more heavily characterized and studied. There are six major enzymes involved in folate synthesis, which, in L.lactis, are contained in five genes: folB, folKE, folP, folC, and folA[1]. The first four, which we have chosen to focus on, are located in a gene cluster approximately 4.4kb long. We’ve chosen not to focus on folA for the time being because folA encodes an enzyme which turns one form of folate (dihydrofolate) into another form of folate(tetrahydrofolate). Since our assay would detect both types of folate as part of the total folate production, folA was not a prime target for overexpression of folate. In previous studies, this folate gene cluster has been successfully transformed into the folate-consuming bacteria L.gasseri, turning the bacteria in to folate-producers[3]. Therefore, we have chosen to also use the folate operon from L.lactis, which also offers the additional benefit of removing the operon from its natural regulatory context.

The folate biosynthesis pathway from L.lactis. [1]
The folate biosynthesis pathway from L.lactis. [1]

Our strategy is to clone the entire folate operon from the L.lactis genome and to transform the entire operon into E.coli. However, because we are unsure of whether or not the ribosomal binding sites (RBS) within the L.lactis operon would work in E.coli, we are also going to unpack the operon by cloning each of the four genes individually, placing them behind E.coli RBSs, and then recombine them into a single empty BioBricks™ plasmid. In addition to the main folate operon, we will also be experimenting with overexpression of the para-aminobenzoic acid (pABA) synthesis pathway from chorismate. Wegkamp et al. have shown that in order to increase overall total levels of folate, both the pABA synthesis genes and certain folate production genes need to be overexpressed[4]. The pABA pathway involves three genes, pabA, pabB, and pabC – though in L.lactis, pabB is actually a fusion gene encoding for both pabB and pabC[4].

System Design

Final folate biosynthesis plasmid
Final folate biosynthesis plasmid
Final folate biosynthesis plasmid
Final folate biosynthesis plasmid

The overall system design for testing folate production in E.coli is to construct two plasmids – one for the folate biosynthesis pathway, and one for the pABA synthesis pathway. In addition to ensuring that the plasmids are complementary, each plasmid would need to contain a different variable copy origin of replication, which would be low copy by default, but can be switched to high copy via the addition of Isopropyl β-D-1-thiogalactopyranoside (IPTG)to the media. This will allow us to test overexpression of each plasmid separately. In addition, each plasmid will contain a constitutive promoter, since we would want folate to be produced constantly. The purple dots represent ribosomal binding sites (RBS), followed by the gene (green arrow), and eventually terminating in a double stop (TAATAA) sequence, as regulated by the Registry of Standard Biological Parts.

Folate Detection Methods

We will be detecting folate production, and thus the relative success of our engineering efforts, via a microbiological assay involving the folate-dependent strain Enterococcus hirae[5]. This assay involves first the characterization of a standard growth curve of E. hirae given known quantities of folate present in the growth media. Once the standard curve has been established, then experimental growth levels, as quantified by spectrophotometry, can be interpolated. PABA concentrations will be measured via high performance liquid chromatography (HPLC) [4].

Folate microbiological assay protocol

para-aminobenzoic acid (pABA) HPLC protocol

Relevant Registry Parts

Basic Parts

Basic Parts (Extracted from L.lactis subspe. IL1403 genome)
Part Name Registry # Description Cloned? Sequence confirmed?
Entire folate synthesis operon BBa_K137002 Includes folB+folKE+folP+ylgG+folC NO NO
folB BBa_K137009 dihydroneopterin aldolase YES YES
folKE BBa_K137011 fusion gene: GTP cyclohydrolase & 2-amino-4-hydroxy-6- hydroxymethyldihydropteridine pyrophosphokinase YES YES
folP BBa_K137012 Dihydropteroate synthase NO NO
folC BBa_K137013 folate synthetase/polyglutamyl folate synthetase NO NO
pabA BBa_K137005 para-aminobenzoate synthetase component II YES YES
pabB BBa_K137006 para-aminobenzoate synthetase component I YES YES

Construction Intermediates

Construction Intermediates: Adding b0034 (strong RBS) before each individual gene
Part Name Registry # Description Cloned? Sequence confirmed?
folB + b0034 BBa_S03957 RBS + dihydroneopterin aldolase YES YES
folKE + b0034 BBa_S03958 RBS + fusion gene: GTP cyclohydrolase & 2-amino-4-hydroxy-6- hydroxymethyldihydropteridine pyrophosphokinase YES YES
folP + b0034 BBa_S03959 RBS + Dihydropteroate synthase NO NO
folC + b0034 BBa_S03960 RBS + folate synthetase/polyglutamyl folate synthetase NO NO
pabA + b0034 BBa_S03976 RBS + para-aminobenzoate synthetase component II YES YES
pabB + b0034 BBa_S03977 RBS + para-aminobenzoate synthetase component I YES YES
b0034 + folB + b0034 + folKE BBa_S03961 Combining folB (with RBS) + folKE (with RBS) YES YES
Construction Intermediates: Adding j23100 (constitutive promoter) to each gene (with RBS already)
Part Name Registry # Description Cloned? Sequence confirmed?
folB + j23100 BBa_S04032 Promoter(j23100) + RBS(b0034) + dihydroneopterin aldolase YES YES
folKE + j23100 BBa_S04033 Promoter + RBS + fusion gene: GTP cyclohydrolase & 2-amino-4-hydroxy-6- hydroxymethyldihydropteridine pyrophosphokinase YES YES
pabA + j23100 BBa_S04034 Promoter + RBS + para-aminobenzoate synthetase component II YES YES
folBKE + j23100 BBa_S04035 Promoter + folB (with RBS) + folKE (with RBS) YES YES
pabB + j23100 BBa_S04039 Promoter + pabB YES YES
pabB + b0015 BBa_S04038 pabB + b0015 terminator MAYBE WAITING

Composite Parts

Composite Parts: Adding b0015 (double terminator) to complete constructs with promoter + RBS already
Part Name Registry # Description Cloned? Sequence confirmed?
folB + b0015 BBa_K137053 Promoter(j23100) + RBS(b0034) + folB (dihydroneopterin aldolase) + double terminator (b0015) YES YES
folKE + b0015 BBa_K137054 Promoter + RBS + folKE (fusion gene: GTP cyclohydrolase & 2-amino-4-hydroxy-6- hydroxymethyldihydropteridine pyrophosphokinase) + double terminator (b0015) NO NO
pabA + b0015 BBa_K137055 Promoter + RBS + pabA (para-aminobenzoate synthetase component II) Maybe waiting
folBKE + b0015 BBa_K137056 Promoter + RBS + folB + folKE + b0015 NO NO

Current Progress

  • 8/01/08
    • On the last cloning step (adding B0015 to everything). Once that's finished I can actually start the metabolic engineering part of the project...

References

  1. Sybesma W, Burgess C, Starrenburg M, van Sinderen D, and Hugenholtz J. . pmid:15113564. PubMed HubMed [sybesma1]
  2. Asrar FM and O'Connor DL. . pmid:16081276. PubMed HubMed [asrar]
  3. Wegkamp A, Starrenburg M, de Vos WM, Hugenholtz J, and Sybesma W. . pmid:15128580. PubMed HubMed [wegkamp1]
  4. Wegkamp A, van Oorschot W, de Vos WM, and Smid EJ. . pmid:17308179. PubMed HubMed [wegkamp2]
  5. Horne DW and Patterson D. . pmid:3141087. PubMed HubMed [horne]
  6. Bernstein JR, Bulter T, and Liao JC. . pmid:18396082. PubMed HubMed [bernstein]
  7. Camilo E, Zimmerman J, Mason JB, Golner B, Russell R, Selhub J, and Rosenberg IH. . pmid:8613033. PubMed HubMed [camilo]
  8. Bermingham A and Derrick JP. . pmid:12111724. PubMed HubMed [bermingham]
  9. Gabelli SB, Bianchet MA, Xu W, Dunn CA, Niu ZD, Amzel LM, and Bessman MJ. . pmid:17698004. PubMed HubMed [gabelli]
  10. Sybesma W, Starrenburg M, Kleerebezem M, Mierau I, de Vos WM, and Hugenholtz J. . pmid:12788700. PubMed HubMed [sybesma2]
  11. Sheng H, Knecht HJ, Kudva IT, and Hovde CJ. . pmid:16885287. PubMed HubMed [sheng]
  12. Morita M, Asami K, Tanji Y, and Unno H. . pmid:11386882. PubMed HubMed [morita]
  13. Yun J, Park J, Park N, Kang S, and Ryu S. . pmid:18051328. PubMed HubMed [yun]
  14. Zhu T, Pan Z, Domagalski N, Koepsel R, Ataai MM, and Domach MM. . pmid:16269750. PubMed HubMed [zhu]
All Medline abstracts: PubMed HubMed
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