Team:WHU-China/Footages/project

From 2014.igem.org

(Difference between revisions)
(Created page with "<p>Formaldehyde is known to be a human carcinogen which do a lot of harm to human beings, especially whose house is newly decorated. And to make matters worse, there are no effec...")
 
Line 1: Line 1:
 +
<html>
 +
<p>Formaldehyde is known to be a human carcinogen which do a lot of
<p>Formaldehyde is known to be a human carcinogen which do a lot of
harm to human beings, especially whose house is newly decorated. And
harm to human beings, especially whose house is newly decorated. And
Line 294: Line 296:
deposition in water. This device can be used in high poisonous
deposition in water. This device can be used in high poisonous
effluence.</p>
effluence.</p>
 +
 +
</html>

Latest revision as of 03:38, 18 October 2014

Formaldehyde is known to be a human carcinogen which do a lot of harm to human beings, especially whose house is newly decorated. And to make matters worse, there are no effective and non-secondary-pollution method to eliminate indoor formaldehyde. To solve this problem, we developed a method of detection and removal of indoor formaldehyde.

Using synthetic biology, we developed a method of detection and removal of indoor formaldehyde, which can solve this problem without secondary-pollution.

Background: About Formaldehyde

Properties

Formaldehyde is the simplest aldehyde. It is a highly reactive gas and is formed by oxidation or incomplete combustion of hydrocarbons. Formaldehyde exists at room temperature as a nearly colorless gas with a pungent, suffocating odor. In its pure state, formaldehyde is not easily handled, because it is extremely reactive and polymerizes readily. The primary form of formaldehyde in dilute aqueous solutions is its monomeric hydrate, methylene glycol (methanediol) and the primary forms in concentrated solutions are oligomers and polymers of polyoxymethylene glycols.[1]

The physical and chemical properties of formaldehyde are summarized in Table 1.

Table 1. Physical and chemical properties of formaldehyde

Formaldehyde is an important precursor to many other materials and chemical compounds. In view of its widespread use, toxicity and volatility, exposure to formaldehyde is a significant consideration for human health. In 2011, the US National Toxicology Program described formaldehyde as "known to be a human carcinogen".[2]

Applications

3.1 Construction and Decorative Products

Since the invention of the particle board in Germany in the 1940s, the use of formaldehyde in glues and resins has revolutionised the construction industry. Formaldehyde-based resins allow wood chips, sawdust and even some recycled wood waste to be combined to create wood products that are durable, cost-effective and high performance alternatives to solid wood. Formaldehyde-based resins also provide better dimensional stability and mould resistance.

Figure 1: Formaldehyde in Construction and Decorative Products

Automotive Applications

Many 'under-the-hood components' such as fuel pumps, transmission parts and brake pads are produced with formaldehyde-based resins. Other formaldehyde based applications include the decorative laminates of car interiors, engine lubricants, vulcanized rubber tyres and lightweight polyurethane foams for automobile door insulation.

Figure 1: Formaldehyde in Construction and Decorative Products

Harm

Toxic effects

Formaldehyde is a highly reactive chemical that causes tissue irritation and damage on contact. Formaldehyde concentrations that have been associated with various toxic effects in humans show wide interindividual variation and are route dependent. Symptoms are rare at concentrations below 0.5 ppm; however, upper airway and eye irritation, changes in odor threshold, and neurophysiological effects (e.g., insomnia, memory loss, mood alterations, nausea, fatigue) have been reported at concentrations ≤ 0.1 ppm. The most commonly reported effects include eye, nose, throat, and skin irritation.

Carcinogenicity

Formaldehyde is known to be a human carcinogen based on sufficient evidence of carcinogenicity from studies in humans and supporting studies on mechanisms of carcinogenesis.

Epidemiological studies have demonstrated a causal relationship between exposure to formaldehyde and cancer in humans. Causality is indicated by consistent findings of increased risks of nasopharyngeal cancer, sinonasal cancer, and myeloid leukemia among individuals with higher measures of exposure to formaldehyde (exposure level or duration), which cannot be explained by chance, bias, or confounding.

Formaldehyde exposure is associated with multiple modes of action related to carcinogenicity, such as DNA reactivity, gene mutation, chromosomal breakage, aneuploidy, epigenetic effects (binding to lysine residues of histones), glutathione depletion, oxidative stress, and cytotoxicity-induced cellular proliferation (Lu et al. 2008, Guyton et al. 2009, NTP 2010). There is evidence for a genotoxic mode of action for formaldehyde-induced cancer; however, the mechanisms by which formaldehyde causes cancer are not completely understood and most likely involve several modes of action.[1]

Figure 1: Formaldehyde in Construction and Decorative Products

To detect and remove the formaldehyde, we design and construct three systems to build our Formaldehyde Terminator.

Coloration system

Since we would like to use formaldehyde as an input in our design,can we find or construct a biobrick sensing the presence of formaldehyde? In Bacillus subtilis 168, there is an operon called hxlAB operon.[1][2]

Figure 1 The formaldehyde promoter shown in the red column

This operon controls the expression of two key enzymes in the ribulose monophosphate pathway that are involved in formaldehyde fixation, 3-hexulose-6-phosphate synthase and 6-phospho-3-hexuloisomerase. [3] Expression of the hxlAB operon is induced by the presence of formaldehyde. HxlR protein can enhance the operon to some extent. The hxlR gene that encodes HxlR protein is located upstream the hxlAB operon.

We get a part of the operon (which we name it P-formaldehyde,shown in a red column in Figure 1) by PCR , add a fluorescent protein or chromoprotein downstream and transform the plasmid(Figure2) into E.coli DH5a. In this way, we can know whether there is formaldehyde or not from the color change.

Figure 2 A GFP reporter is added downstream of the formaldehyde promoter

The street cleaner system

PADH, formaldehyde dehydrogenase from Pseudomonas putida is a formaldehyde dehydrogenase which can oxidize free formaldehyde to formate independent of GSH. [4] FDH, formate dehydrogenase from Candida boidinii can oxidize formate to carbon dioxide in the presence of NAD+.[5][6]

Figure 3 The mechanism of two enzymatic reactions

We would like to use this two enzymes to degrade formaldehyde into water and carbon dioxide. In our design,the expression of this two enzymes can be controlled by the P-formaldehyde so that the presence of formaldehyde can induce the expression of them. We can also use constitutive promoter to guarantee the expression level.

Figure 4 Two enzymes are added downstream of the formaldehyde promoter

lysis system

Lysis system has two important roles in our design. One is about biosafety; the other is to help the enzymes come out of the bacteria so that they can work better in the environment.

We use Lux system, E7 protein and our P-formaldehyde to construct this system.

Lux system is a quorum sensing system , consisting of luxI, luxR and P-lux. luxI is a synthase for a small molecule called acyl-homoserine lactone (AHL). AHL can bind to the LuxR protein and then the composite can stimulate transcription from the right hand lux promoter P-lux.[7]

Figure 5 The mechanism of the lux system

The E7 lysis protein is a key component of the SOS response system in E.coli and functions to export bacteriocins into the extracelluar space under stressful environmental conditions. Besides, this protein only has 47 amino acids and thus can be easily utilized as an modular part in a constructed biobrick which helps the expressed foreign protein easier to be collected or take effect outside cell.[8][9] In this case, E7 lysis protein is used to release enzyme FDH and enzyme PADH to the extracellular space so that they can degrade formaldehyde in a more efficient way.We design this system as following:

Figure 6 A system which can release two enzymes out of the cells when stimulated by formaldehyde

When the P-formaldehyde is activated, the bacteria will express FDH, PADH and LuxI protein. LuxI synthesis AHL and AHL binds to LuxR so that the P-lux is activated and the E7 protein is expressed. When E7 reaches a certain level, the bacteria lyse.

reference

  • [1] Yurimoto H, Hirai R, Matsuno N, et al. HxlR, a member of the DUF24 protein family, is a DNA© binding protein that acts as a positive regulator of the formaldehyde©inducible hxlAB operon in Bacillus subtilis[J]. Molecular microbiology, 2005, 57(2): 511-519.
  • [2] Huyen N T T, Eiamphungporn W, Mäder U, et al. Genome© wide responses to carbonyl electrophiles in Bacillus subtilis: control of the thiol© dependent formaldehyde dehydrogenase AdhA and cysteine proteinase YraA by the MerR© family regulator YraB (AdhR)[J]. Molecular microbiology, 2009, 71(4): 876-894.
  • [3] Yasueda H, Kawahara Y, Sugimoto S. Bacillus subtilis yckG andyckF Encode Two Key Enzymes of the Ribulose Monophosphate Pathway Used by Methylotrophs, and yckH Is Required for Their Expression[J]. Journal of bacteriology, 1999, 181(23): 7154-7160.
  • [4] Ito K, Takahashi M, Yoshimoto T, et al. Cloning and high-level expression of the glutathione-independent formaldehyde dehydrogenase gene from Pseudomonas putida[J]. Journal of bacteriology, 1994, 176(9): 2483-2491.
  • [5] Sheng B, Zheng Z, Lv M, et al. Efficient Production of (R)-2-Hydroxy-4-Phenylbutyric Acid by Using a Coupled Reconstructed d-Lactate Dehydrogenase and Formate Dehydrogenase System[J]. PloS one, 2014, 9(8): e104204.
  • [6] Khangulov S V, Gladyshev V N, Dismukes G C, et al. Selenium-containing formate dehydrogenase H from Escherichia coli: a molybdopterin enzyme that catalyzes formate oxidation without oxygen transfer[J]. Biochemistry, 1998, 37(10): 3518-3528.
  • [7] Winson M K, Camara M, Latifi A, et al. Multiple N-acyl-L-homoserine lactone signal molecules regulate production of virulence determinants and secondary metabolites in Pseudomonas aeruginosa[J]. Proceedings of the National Academy of Sciences, 1995, 92(20): 9427-9431.
  • [8]Lin L J R, Liao C C, Chen Y R, et al. Induction of membrane permeability in Escherichia coli mediated by lysis protein of the ColE7 operon[J]. FEMS microbiology letters, 2009, 298(1): 85-92.
  • [9] Chak K F, Kuo W S, James R. Cloning and characterization of the ColE7 plasmid[J]. Journal of general microbiology, 1991, 137(1): 91-100.

Application:

To make our detection and street cleaner systems more convenient and functional for others to use in both daily life and industry (this is also a requirement for a good iGEM project), we have made a device (see "A Device for Daily Life") and got a detailed plan about industrial application (see "Devices for Industrial Application").

A Device for Daily Life

We have made a convenient device(showed below) to hold our E.coli so that we can detect the formaldehyde gas easier. A electronic formaldehyde detector was added so that we can compare our detection system with it.

In our future work, we will combine this device with automatic recording machine. By connecting them, we can get data automatically and precisely. Then we will optimize it by diminishing its volume and embellishing its appearance so that it can be easier to use.

Devices for Industrial Application

Although there still remains a lot for our project to come to be used practically, we can hope that one day our dream can come true. As we know from the information above, our systems can be used separately; one way is that our Street Cleaner System can help to clean formaldehyde from industrial effluent. But if we want to use it in a higher efficient way, we may make use of engineering machine. Here we show two possible devices:

The first one is Bacterial Board, we can see from DEVICE I.

This device is mainly composed by two semi-permeable membranes and a container that contains bacteria, this device is inclined against water direction, and this can limit the influence of deposition in effluent to semi-membrane. Bacteria in container is access to culture jar, it can keep flowing and provide bacteria with successive nutrient and oxygen. It can be used to effluent that has low poison. After setting series of these devices in an area of sewage duct, we can clean formaldehyde efficiently.

The second one is bacterial windmill, we can see from DEVICE II,

The windmill has a lot of containers, these containers contain bacteria, when the windmill rotates toward water direction, they can contact effluent one by one, and then it can be above the surface of effluent, it provides bacteria with chance to get both formaldehyde and oxygen, bacteria can also avoid being submerged in harmful effluence for long time, and it can also limit the influence of deposition in water. This device can be used in high poisonous effluence.