Team:Caltech/Project/Vitamins

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==Folate Detection Methods==
==Folate Detection Methods==
[[Image:Lrham.jpg| thumb| left| 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].]]
[[Image:Lrham.jpg| thumb| left| 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].]]
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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 ''Lactobacillus rhamnosus''<cite>horne</cite>. The microbiological assay involves growing up test cultures of ''E. coli,'' centrifuging and lysing the cultures, separating the supernatant from the lysate, inoculating each sample with ''L. rhamnosus'', and measuring the relative growth of each sample compared to controls at 546nm using a plate reader. In order to quantify the relative growth of the folate-dependent strain ''L. rhamnosus'', a standard growth curve must first be characterized using known quantities of folic acid in water. Once the standard curve has been established, then experimental growth levels, as quantified by spectrophotometry, can be interpolated.  
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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 ''Lactobacillus rhamnosus''<cite>horne</cite>. The microbiological assay involves growing up test cultures of ''E. coli,'' centrifuging and lysing the cultures, separating the supernatant from the lysate, inoculating each sample with ''L. rhamnosus'', and measuring the relative growth of each sample compared to controls at 546nm using a plate reader. Since the assay media specifically lacks folate, it becomes the limiting factor in the growth of ''L. rhamnosus'', with the only possible source being the bacterial lysates of the engineered ''E. coli''. In order to quantify the relative growth of the folate-dependent strain ''L. rhamnosus'', a standard growth curve must first be characterized using known quantities of folic acid in water. Once the standard curve has been established, then experimental growth levels, as quantified by spectrophotometry, can be interpolated.  
[[Team:Caltech/Protocols/Folate assay|Folate Microbiological Assay Protocol]]  
[[Team:Caltech/Protocols/Folate assay|Folate Microbiological Assay Protocol]]  

Revision as of 13:09, 18 September 2008


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In vivo Folate Production


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 (derived from GTP), p-aminobenzoic acid (pABA, derived from chorismate), and a poly-glutamyl tail (derived from linking 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. The microbiological assay involves growing up test cultures of E. coli, centrifuging and lysing the cultures, separating the supernatant from the lysate, inoculating each sample with L. rhamnosus, and measuring the relative growth of each sample compared to controls at 546nm using a plate reader. Since the assay media specifically lacks folate, it becomes the limiting factor in the growth of L. rhamnosus, with the only possible source being the bacterial lysates of the engineered E. coli. In order to quantify the relative growth of the folate-dependent strain L. rhamnosus, a standard growth curve must first be characterized using known quantities of folic acid in water. Once the standard curve has been established, then experimental growth levels, as quantified by spectrophotometry, can be interpolated.

Folate Microbiological Assay Protocol

pABA Detection Methods

para-Aminobenzoic Acid [http://en.wikipedia.org/wiki/4-Aminobenzoic_acid]

para-Aminobenzoic Acid (pABA) can be detected using high performance liquid chromatography (HPLC). Using a 14 minute protocol (see link below), we were able to detect pABA peaks coming off a C18 column at 4.9 min. A standard curve was made by running a series of 1:3 pABA dilutions starting at a 10ug/ml concentration. The pABA for the standard curve was spiked into wild type E. coli cell lysate, which by itself did not show any detectable pABA peaks. Samples were run using the same HPLC protocol as the standards, and both lysate and supernatant were tested for pABA peaks.

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 able to successfully extract and clone the following four genes: folB, folKE, pabA, and pabB. We 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.

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.

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.

Folate Assay Results for folB, folKE, and folBKE

The experimental setup included running a standard curve with known amounts of folic acid (0-10ng) simultaneously with the samples, in order to have the same basis for comparison. The hope was that the standard curve growth OD values would correspond with known concentrations, and so sample concentrations could be interpolated based upon the standard. For folKE and folBKE, which were in constitutive high-copy plasmids, these genes were tested with and without the addition of 500 ng of pABA during inoculation. folB, which is in an inducible copy plasmid, it was tested induced high-copy as well as uninduced low-copy, with and without 500 ng of pABA during inoculation. Folate levels were assayed based on protocol mentioned above[1]. We assayed both the supernatant and the cell lysate, though only the supernatant had measurable results. As such, the following data contain only supernatant assay results.

For Test #1, only folKE and folBKE were tested, as folB needed a longer incubation time. The standard curve and growth results are shown on the right. First off, it is clear that the standard curve is pretty much useless in terms of linearity and relative growth compared to the samples. Although the reasons for this were unclear, the growth data for the samples are very encouraging since the relative folate levels match what we would expect. We see that the addition of 500 ng of pABA during inoculation dramatically increases overall folate levels for both folKE and folBKE relative to the samples without pABA. If folKE and folBKE folate levels are compared, it appears that overexpression of only folKE with pABA produces more folate than overexpression of both folB and folKE. Furthermore, adding pABA to the wild type controls did not affect growth at all, suggesting that the assay bacteria L. rhamnosus was not metabolizing the extra pABA. The extremely high OD values for all the samples were possibly the result of not completely washing out all of the culture media prior to adding the L. rhamnosus to the assay samples.

When we repeated the assay in duplicate(above figures), this time on folB as well, we were able to observe the same trends in growth with and without the addition of pABA. In addition, the standard curve came out perfectly linear in the expected range from .1 - 1ng. However, the growth OD for the standard curve again did not correspond with sample growth, and so we were again unable to estimate the folate concentrations in our samples.

Increasing the flux of both pathways upstream of pABA integration into the pterin (derived from GTP) component of folate may have created a bottleneck. Unfortunately we were unable to test this hypothesis since we could not overexpress folP.

Again, we see that for folKE and folBKE, the addition of pABA does increase total folate levels, though not as dramatically as before. Overexpression of folKE alone with pABA continues to produce more folate than overexpression of folBKE. The rampant growth of the wild type control is disconcerting, but wild type folate production again appears to be unaffected by the addition of pABA. Furthermore, folate levels for folKE and folBKE are still higher than the control where L. rhamnosus was added to only media without supernatant.

And what of folB? Recall that folB was the only gene to be successfully cloned into an inducible-copy plasmid, and so it was tested both induced and uninduced. The folate levels in the induced sample (high-copy) are higher than in the uninduced (low-copy) sample, which is consistent with what we would expect. The addition of pABA to both induced and uninduced increases relative levels of folate, which is also consistent. However, the interesting thing to note is that folate levels for the +pABA samples are the same for induced and uninduced. Of course, given the small sample size, this could just be due to variability, but it could also suggest that folate production hit an upper rail. This explanation seems even more likely if we reconsider the folate biosynthesis pathway (shown again to the right), and we see that folB and folKE are both located upstream of actual integration of pABA into the molecule, accomplished by folP. It is very possible that we are increasing the flux of both pathways going into the folP junction, but as we were unable to overexpress folP as well, we may have created a bottleneck.

para-Aminobenzoic Acid (pABA) HPLC Assay Results for pabA and pabB

Using high performance liquid chromatography (HPLC) we were able to detect pABA peaks from overexpression of pabA and pabB individually compared to a control sample of wild type supernatant. Compared to the peak areas of the standard curve, we were able to use a linear approximation to determine the concentration of pABA to be 703.2 ng/mL for pabA overexpression, and 805.9 ng/mL for pabB.

Conclusions and Future Work

We have shown, through very preliminary tests, the feasibility of overproduction of folate in E.coli using genes originally derived from L.lactis. Our data have confirmed previous studies in the necessity of overexpressing both folate and pABA synthesis genes, and we have shown that folate appears to be mostly present in the supernatant. Our data also suggest that overexpression of folKE is the most effective, and in fact, more effective than overexpressing folB + folKE.

We have also shown that it is possible to overexpress para-aminobenzoic acid production in E. coli and that overexpression of either pabA or pabB increases total levels of para-aminobenzoic acid (pABA).

Further work on this project would include repeating the folate assay to generate more data, perfecting the assay in order to quantify folate levels with the standard curve, making the pabA + pabB construct, making a folB + folKE + pabA + pabB construct, and extracting and cloning folP from the L.lactis genome.

Relevant 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>

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