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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 --> future link here when i figure out how to make a new page.
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[[Folate microbiological assay protocol]]
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[[para-aminobenzoic acid (pABA) HPLC protocol]]
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==Relevant Registry Parts==
 
==Relevant Registry Parts==
 
===Basic Parts===
 
===Basic Parts===
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==Current Progress==
 
==Current Progress==
*7/10/08  
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*8/01/08  
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**On the last cloning step (adding B0015 to everything). Once that's finished I can actually start the metabolic engineering part of the project...
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* [[Media:Vhsiao_Abstract_Final_V3.pdf |Abstract for SFP Summer Session]]
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* [[Media:Amgen_Progress_Report_1.pdf | Progress Report #1]]
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* [[Media:Final_Amgen_Progress_Report_2.pdf | Progress Report #2]]
  
 
==References==
 
==References==

Latest revision as of 15:52, 1 August 2008


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iGEM 2008

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Vitamin production

Background Information on Folate

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]

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]

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

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. Multivitamin production in Lactococcus lactis using metabolic engineering. Metab Eng. 2004 Apr;6(2):109-15. DOI:10.1016/j.ymben.2003.11.002 | PubMed ID:15113564 | HubMed [sybesma1]
  2. Asrar FM and O'Connor DL. Bacterially synthesized folate and supplemental folic acid are absorbed across the large intestine of piglets. J Nutr Biochem. 2005 Oct;16(10):587-93. DOI:10.1016/j.jnutbio.2005.02.006 | PubMed ID:16081276 | HubMed [asrar]
  3. Wegkamp A, Starrenburg M, de Vos WM, Hugenholtz J, and Sybesma W. Transformation of folate-consuming Lactobacillus gasseri into a folate producer. Appl Environ Microbiol. 2004 May;70(5):3146-8. PubMed ID:15128580 | HubMed [wegkamp1]
  4. Wegkamp A, van Oorschot W, de Vos WM, and Smid EJ. Characterization of the role of para-aminobenzoic acid biosynthesis in folate production by Lactococcus lactis. Appl Environ Microbiol. 2007 Apr;73(8):2673-81. DOI:10.1128/AEM.02174-06 | PubMed ID:17308179 | HubMed [wegkamp2]
  5. Horne DW and Patterson D. Lactobacillus casei microbiological assay of folic acid derivatives in 96-well microtiter plates. Clin Chem. 1988 Nov;34(11):2357-9. PubMed ID:3141087 | HubMed [horne]
  6. Bernstein JR, Bulter T, and Liao JC. Transfer of the high-GC cyclohexane carboxylate degradation pathway from Rhodopseudomonas palustris to Escherichia coli for production of biotin. Metab Eng. 2008 May-Jul;10(3-4):131-40. DOI:10.1016/j.ymben.2008.02.001 | PubMed ID:18396082 | HubMed [bernstein]
  7. Camilo E, Zimmerman J, Mason JB, Golner B, Russell R, Selhub J, and Rosenberg IH. Folate synthesized by bacteria in the human upper small intestine is assimilated by the host. Gastroenterology. 1996 Apr;110(4):991-8. PubMed ID:8613033 | HubMed [camilo]
  8. Bermingham A and Derrick JP. The folic acid biosynthesis pathway in bacteria: evaluation of potential for antibacterial drug discovery. Bioessays. 2002 Jul;24(7):637-48. DOI:10.1002/bies.10114 | PubMed ID:12111724 | HubMed [bermingham]
  9. Gabelli SB, Bianchet MA, Xu W, Dunn CA, Niu ZD, Amzel LM, and Bessman MJ. Structure and function of the E. coli dihydroneopterin triphosphate pyrophosphatase: a Nudix enzyme involved in folate biosynthesis. Structure. 2007 Aug;15(8):1014-22. DOI:10.1016/j.str.2007.06.018 | PubMed ID:17698004 | HubMed [gabelli]
  10. Sybesma W, Starrenburg M, Kleerebezem M, Mierau I, de Vos WM, and Hugenholtz J. Increased production of folate by metabolic engineering of Lactococcus lactis. Appl Environ Microbiol. 2003 Jun;69(6):3069-76. PubMed ID:12788700 | HubMed [sybesma2]
  11. Sheng H, Knecht HJ, Kudva IT, and Hovde CJ. Application of bacteriophages to control intestinal Escherichia coli O157:H7 levels in ruminants. Appl Environ Microbiol. 2006 Aug;72(8):5359-66. DOI:10.1128/AEM.00099-06 | PubMed ID:16885287 | HubMed [sheng]
  12. Morita M, Asami K, Tanji Y, and Unno H. Programmed Escherichia coli cell lysis by expression of cloned T4 phage lysis genes. Biotechnol Prog. 2001 May-Jun;17(3):573-6. DOI:10.1021/bp010018t | PubMed ID:11386882 | HubMed [morita]
  13. Yun J, Park J, Park N, Kang S, and Ryu S. Development of a novel vector system for programmed cell lysis in Escherichia coli. J Microbiol Biotechnol. 2007 Jul;17(7):1162-8. PubMed ID:18051328 | HubMed [yun]
  14. Zhu T, Pan Z, Domagalski N, Koepsel R, Ataai MM, and Domach MM. Engineering of Bacillus subtilis for enhanced total synthesis of folic acid. Appl Environ Microbiol. 2005 Nov;71(11):7122-9. DOI:10.1128/AEM.71.11.7122-7129.2005 | PubMed ID:16269750 | HubMed [zhu]
All Medline abstracts: PubMed | HubMed