Team:Caltech/Project/Vitamins

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Contents

Background Information on Folate

The structure of tetrahydrofolate sybesma1

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 sybesma1. 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 intestineasrar.

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. sybesma1

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 folAsybesma1. 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-producerswegkamp1. 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. sybesma1

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 overexpressedwegkamp2. 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 pabCwegkamp2.

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

Image of Lactobacillus rhamnosus. Itself a probiotic strain, L.rhamnosus is commonly used in yogurt [http://www.yaklasansaat.com/resimler/2008haberresimleri/haber_subat_resim/subat19_lactobacillus_rhamnosus.jpg][http://en.wikipedia.org/wiki/Lactobacillus_rhamnosus].

We will be detecting folate production, and thus the relative success of our engineering efforts, via a microbiological assay involving the folate-dependent strain Lactobacillus rhamnosushorne. This assay involves first the characterization of a standard growth curve of L. rhamnosus 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) wegkamp2.

Folate Microbiological Assay Protocol

para-aminobenzoic acid (pABA) HPLC protocol

Results

Modifications to System Design

Target constructs for folate biosynthesis and pABA synthesis gene overexpression. We were able to successfully clone folB in an inducible-copy plasmid and folKE, pabA, pabB and folBKE in high-copy plasmids. Unfortunately, we were unable to complete the pabA + pabB construct as of August 23rd.

We were unable to successfully use PCR to clone out several of our goals: the entire folate gene cluster, folP, and folC. After analyzing the sequencing results of our other folate biosynthesis genes, we realized that the genetic sequences had several point mutations which indicated that we had a homologous, but not identical, genomic DNA than the one that we had based our PCR primer design off of. We believe that this homologous sequence could be the reason why we were unable to successfully extract folP, folC, and subsequently, the entire folate gene cluster (since folP and folC are the last two genes in the cluster) from the genomic DNA of L. lactis that we had ordered from ATCC. The revised target constructs are shown in the figure to the right.

As a result, we continued forward with cloning for the four genes we were able to successfully extract: folB, folKE, pabA, and pabB. We still aimed to create individual constructs of each gene in the IPTG inducible-copy plasmid pSB2K3, as well as constructs with the two folate genes combined and with the two pABA synthesis genes combined. However, we had issues cloning our genes into the pSB2K3 + B0015 terminator construct, and so switched to completing the constructs in the high copy plasmid pSB1AK3 + b0015 terminator.

As such, our final constructs are: folB in pSB2K3, and folKE, pabA, folB+folKE, and pabB in pSB1AK3. Although the last two were not yet sequence confirmed, we pushed ahead to testing due to time constraints. The goal was to detect higher folate levels with overexpression of folB, folKE, and folBKE with pABA spiked in. Previous studies on folate overexpression have shown that both folate synthesis and pABA synthesis genes need to be overexpressed simultaneously in order to increase total folate levels Wegkamp. As such, tests were done with and without the spiking in of pABA during the E.coli inoculation.

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 [http://partsregistry.org/Part:BBa_K137002 BBa_K137002] Includes folB+folKE+folP+ylgG+folC NO NO
folB [http://partsregistry.org/Part:BBa_K137009 BBa_K137009] dihydroneopterin aldolase YES YES
folKE [http://partsregistry.org/Part:BBa_K137011 BBa_K137011] fusion gene: GTP cyclohydrolase & 2-amino-4-hydroxy-6- hydroxymethyldihydropteridine pyrophosphokinase YES YES
folP [http://partsregistry.org/Part:BBa_K137012 BBa_K137012] Dihydropteroate synthase NO NO
folC [http://partsregistry.org/Part:BBa_K137013 BBa_K137013] folate synthetase/polyglutamyl folate synthetase NO NO
pabA [http://partsregistry.org/Part:BBa_K137005 BBa_K137005] para-aminobenzoate synthetase component II YES YES
pabB [http://partsregistry.org/Part:BBa_K137006 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 [http://partsregistry.org/Part:BBa_S03957 BBa_S03957] RBS + dihydroneopterin aldolase YES YES
folKE + b0034 [http://partsregistry.org/Part:BBa_S03958 BBa_S03958] RBS + fusion gene: GTP cyclohydrolase & 2-amino-4-hydroxy-6- hydroxymethyldihydropteridine pyrophosphokinase YES YES
folP + b0034 [http://partsregistry.org/Part:BBa_S03959 BBa_S03959] RBS + Dihydropteroate synthase NO NO
folC + b0034 [http://partsregistry.org/Part:BBa_S03960 BBa_S03960] RBS + folate synthetase/polyglutamyl folate synthetase NO NO
pabA + b0034 [http://partsregistry.org/Part:BBa_S03976 BBa_S03976] RBS + para-aminobenzoate synthetase component II YES YES
pabB + b0034 [http://partsregistry.org/Part:BBa_S03977 BBa_S03977] RBS + para-aminobenzoate synthetase component I YES YES
b0034 + folB + b0034 + folKE [http://partsregistry.org/Part:BBa_S03961 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 [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

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) YES YES
pabA + b0015 [http://partsregistry.org/Part:BBa_K137055 BBa_K137055] Promoter + RBS + pabA (para-aminobenzoate synthetase component II) YES YES
pabB + b0015 [http://partsregistry.org/Part:BBa_K137109 BBa_K137109] Promoter + RBS + pabB (para-aminobenzoate synthetase component I) YES NO
folBKE + b0015 [http://partsregistry.org/Part:BBa_K137056 BBa_K137056] Promoter + RBS + folB + folKE + b0015 YES NO

References

<biblio>

  1. bernstein pmid=18396082
  2. camilo pmid=8613033
  3. asrar pmid=16081276
  4. bermingham pmid=12111724
  5. gabelli pmid=17698004
  6. sybesma1 pmid=15113564
  7. sybesma2 pmid=12788700
  8. sheng pmid=16885287
  9. morita pmid=11386882
  10. yun pmid=18051328
  11. zhu pmid=16269750
  12. wegkamp1 pmid=15128580
  13. wegkamp2 pmid=17308179
  14. horne pmid=3141087

</biblio>