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From 2011.igem.org

Biobrick: Phosphodiesterase (PDE)

Host:
E.coli Top 10

Origin:
Amplified from the Pseudomonas aeruginosa PAO1 genome

Function & Use:
Phosphodiesterases are a family of enzymes naturally present in microorganisms, which break phosphodiester bonds. The specific enzyme we are using breaks the phosphodiester bond in the second messenger nucleotide, cyclic digaunylate (cyclic-di-GMP). Cyclic-di-GMP is important in many bacterial processes, including biofillm formation and motility. The enzyme regulates signal transduction by controlling levels of the signalling molecule in cells.

We are using the enzyme to control the levels of cyclic-di-GMP within the cell. We expect that the targeted expression of phosphodesiterase could be used to interfere with biofilm formation or to trigger dispersal.

Safety:
Phosphodiesterase enzymes are naturally present in many organisms, including E.coli. We are using it to disrupt biofilm formation by disrupting levels of cyclic-di-GMP, which is the natural function of the enzyme and has already been studied. We do not anticipate the phosphodiesterase biobrick posing a safety risk.

References & Further Reading:

"The EAL Domain Protein VieA Is a Cyclic Diguanylate Phosphodiesterase" which can be accessed here here.

"A blue light-inducible phosphodiesterase activity in the cyanobacterium Synechococcus elongates" which can be accessed here.  

Biobrick: Phosphodiesterase with 6xHIS Tag

We have also created a version of the phosphodiesterase biobrick with 6xHIS tag for affinity purification.

The use of 6xHIS tags is well established and would not give the microorganism any enhanced ability to survive outside the lab, or pose a safety risk to the public or environment.

References & Further Reading:

  Information on "His-tagged proteins", which can be accessed here.

Information on "Affinity Purification", which can be accessed here.

Biobrick: LOV2

Host:
E.coli (Top 10)

Origin:
The LOV domain was originally isolated from Arabidopsis. It was characterised by Dr John Christie of the University of Glasgow.

The version of LOV2 we are using was codon optimised for expression in E.coli and synthesized by researchers from the University of Glasgow’s Institute Of Infection, Immunity and Inflammation.

Function & Uses:
The LOV (Light-Oxygen-Voltage) domain is a photoreceptor that responds to blue light. In nature it was first found to be involved in the phototropism response in plants and has since been found to be present in fungi and bacteria also. It has been shown to be coupled to many domains, for example phosphodiesterase or kinases.

We are using it in our cells as a reporter due to its ability to function in anoxic conditions. This is particularly useful in biofilms and is a function that fluorescent proteins derived from GFP do not have.

Safety:
It has not been attached to any functional domain and the LOV domain itself has no pathogenic properties. We therefore do not expect the LOV2 domain to have any safety risks.

It has previously been expressed in E.coli by Dr John Christie.

References & Further Reading:

"LOV (light, oxygen, or voltage) domains of the blue-light photoreceptor phototropin (nph1): Binding sites for the chromophore flavin mononucleotide" which can be accessed here.

"The LOV Domain Family: Photoresponsive Signaling Modules Coupled to Diverse Output Domains" which can be accessed here.

"Information on the LOV Domain" by Dr John Christie which can be accessed here.
 

Biobrick: LOV2 domain with 6xHIS Tag

We have also created a version of the LOV2 biobrick with a 6xHIS tag for affinity purification.

The use of 6xHIS tags is well established and would not give the microorganism any enhanced ability to survive outside the lab, or pose a safety risk to the public or environment.

References & Further Reading

  Information on "His-tagged proteins", which can be accessed here.

Information on "Affinity Purification", which can be accessed here.

Biobrick: iLOV

Host:
E.coli (Top 10)

Origin:
iLOV is a version of LOV2 that has been altered through site directed mutagenesis and DNA shuffling.

Function & Use
iLOV has the same function and uses as LOV2. As a reporter, it is advantageous over GFP derived fluorescent proteins due to its small size, ability to recover quickly from photobleaching and use in anoxic conditions.

Safety:
The mutations that iLOV has the function of increasing intensity of fluorescence only. It does not have any altered biological role or new biological function.

Therefore we do not antipate any way in which it could increase pathogenicity or survival in the wild.
References and Further Reading:

"The photoreversible fluorescent protein iLOV outperforms GFP as a reporter of plant virus infection" which can be accessed here.  

Biobrick: Ranaspumin (RSN2)

Host:
E.coli (Top 10)

Origin:
Túngara Frog (Engystomops pustulosus)

Function & Uses:
Ranaspumin is a surfactant protein found in the foam nests of the Túngara frog. In nature it is used to protect and incubate the fertilized eggs of the frog. It has natural antimicrobial functions and antibiofilm activity has been reported.

Safety:
We are using it to aid in dispersal of biofilms, which is a natural property of the protein. It has no known biological risk to humans or the environment.

References & Further Reading
"Ranaspumin-2: Structure and Function of a Surfactant Protein from the Foam Nests of a Tropical Frog" which can be accessed here.

"Biofoams and natural protein surfactants" which can be accessed here.  

Biobrick: Ranaspumin with 6xHIS tag

We have also created a version of the Ranaspumin biobrick with a 6xHIS tag.

The HIS tag would not give the microorganism any enhanced ability to survive outside the lab, or pose a safety risk to the public or environment.

References & Further Reading

  Information on "His-tagged proteins", which can be accessed here.

Information on "Affinity Purification", which can be accessed here.  

Biobrick: Latherin

Host: E.coli Top 10

Origin: Horse (Equus caballas)

Function & Uses:
Latherin is a surfactant protein that was originally isolated from horse sweat. Its normal biological function is temperature regulation and is believed to function by enhancing evaporation from the pelt. Due to its ability to bind to hydrophobic surfaces, the protein is being investigated for its possible ability to aid in breaking up biofilms.

Safety:
We are using latherin to aid in dispersal of biofilms, which is a natural property of the protein that has already been investigated.

Latherin is suspected to be involved in the allergen response to horses. We propose that anything containing the protein should be clearly labelled as such, so that those with a horse allergy can avoid contact with it.

References & Further Reading:

"Latherin: A Surfactant Protein of Horse Sweat and Saliva" which can be accessed here

"Isolation and characterization of latherin, a surface-active protein from horse sweat" which can be accessed here.

"Biochemical characterization and surfactant properties of horse allergens" which can be accessed here.

"Latherin and other biocompatible surfactant proteins" which can be accessed here

Biobrick: Latherin with 6xHIS tag

We have also created a version of the Latherin biobrick with 6xHIS tag for affinity purification.

The use of 6xHIS tags is well established and would not give the microorganism any enhanced ability to survive outside the lab, or pose a safety risk to the public or environment.

References & Further Reading

  Information on "His-tagged proteins", which can be accessed here.

Information on "Affinity Purification", which can be accessed here.  

Nissle 1917 Strain of E.coli

Nissle 1917 is a strain of E.coli that we are working with due to its ability to form biofilms. This is a capacity that laboratory strains of E.coli have lost.

It is used as a probiotic in health supplements, which are available to buy commercially in Germany as Mutaflor tablets. It is also studied as a carrier for therapeutic molecules, due to the fact it is safe for human consumption and does not cause illness when it colonises the gut.

References & Further Reading:
"Intestinal immunity of Escherichia coli NISSLE 1917: a safe carrier for therapeutic molecules" which can be accessed here.

"Maintaining remission of ulcerative colitis with the probiotic Escherichia coli Nissle 1917 is as effective as with standard mesalazine" which can be accessed here.

"NF- B- and AP-1-Mediated Induction of Human Beta Defensin-2 in Intestinal Epithelial Cells by Escherichia coli Nissle 1917: a Novel Effect of a Probiotic Bacterium" which can be accessed here.