BacMan

Introducing to you BacMan, protagonist of Team IISER Kolkata’s project. BacMan is an engineered bacterium designed to uptake and sequester arsenic from the ingested food inside the gut at the prevailing physiological conditions. The bacteria will be delivered through a probiotic pill into the GI tract. Probiotic treatment of Arsenicosis with engineered bacteria beneficial and suitable to colonize the gut presents a novel prevention based approach to combat the disease. The bacteria upon serving their purpose will be flushed out from the body by the innate excretory system along with faeces.

The primary aim of Team IISER Kolkata is to establish the proof of concept in the easy to work with model organism E. coli and then proceed to translate the results to a probiotic species such as Lactobacillus spp.

BacMan’s Superpower and Toolset

In order to successfully accomplish the desired purpose, BacMan has to have a set of diverse tools:

1. Innate Arsenic Resistance Mechanism of the Bacteria
   Most bacterial species have an arsenic efflux system that makes them resistant to it as all of the uptaken ions are actively transported out of the cell. Allowing wild type functioning of this efflux system is discouraging to our ambitions of designing a bacteria that can uptake and keep the arsenic ions sequestered within. Hence, the genes coding for this efflux pathway need to be deleted.
2. Arsenic sensing unit
   The bacterium must be able to sense to Arsenic concentration outside and inside the cell so that it can launch a response to uptake and sequester it only when substantially high amounts of the toxic ion are present. A constitutively on response from the bacteria will be an extra stress for the cellular metabolism of the microbe.
3. Arsenic responsive unit
   Upon sensing arsenic concentrations beyond threshold, the bacteria must be able to switch on its response circuit.
4. Arsenic uptake channels and transporters
   The bacterium must be able uptake arsenic once ample amounts are sensed in the surrounding.
5. Arsenic sequestration response
   Once the arsenic starts pouring into the bacterial cell and it’s response circuitry is switched on, an arsenic sequestering protein must be produced to chelate the heavy metal ions.
6. Bacterial recovery unit
   Binding of ligand to any protein is a reversible process and as the sequestering proteins start getting saturated within the cell, the backward reaction will be favored and chelated arsenate and arsenite ions will dissociate and diffuse freely within the cell. These ions are toxic as they bind nonspecifically to several important enzymes and proteins involved in bacterial metabolism thereby disrupting their natural function. Therefore, there has to be a system within the bacterial cell that will recover it from the saturation reached and keep pushing the equilibrium towards the forward direction making the sequestration biphasic.

Detailed explanation of the components of the BacMan

1. Verifying presence of and thereafter deleting ars Operon
   Most bacteria have a chromosomal or plasmid encoded arsenic resistance operon arsRDABC. arsC converts As(V) in the cell to As(III). Genes arsA and arsB of the operon code for an ATP driven arsenite pump that then extrude As(III) out of the cell. arsR and arsD are regulatory proteins that control the expression of the operon.
   
   Ref: The ars Operon of Escherichia coli Confers Arsenical and Antimonial Resistance, Carlin et. al., Journal of Bacteriology, 1995
   
   This resistance system encoded by the operon effluxes out the arsenic ions and is hence undesirable in a bacterial strain being designed to to sequester arsenite and arsenate. Therefore, the primary requirement is to verify the presence of ars operon and if present, delete it.
2. Arsenic channels and transporters
   Arsenic exists in two oxidation states As(III) and As(V). Pentavalent As(V) exists as an oxyanion AsO4 3- which is chemically very similar to phosphate and hence is taken up nonspecifically through Pst and Pit and phosphate transporters in bacterial cells. Trivalent As(III) is found as neutral hydroxide As(OH)3 which structurally mimics Glycerol. Thus AQP channels such as GlpF membrane protein that are actually conduits with large pores that facilitate bidirectional diffusion of water, glycerol and other neutral solutes also allow As(OH)3 through them.
   
   Ref: Pathways of Arsenic Uptake and Efflux, H.C. Yang et. al., Curr. Topics in Membranes, 2015
   
   The above image depicts the mechanism of action of ars Operon which imparts arsenic resistance to the bacterial cell by causing its efflux. Also shown are the pathways of arsenate and arsenite uptake by the cell through phosphate channels and GlpF aquaporins.
3. Arsenic Sensing and Responsive Unit
   After we delete the innate efflux mechanism and enhance arsenic uptake through above mentioned pathways, we plan to create a genetic circuitry that will respond to the building internal concentration of arsenite and arsenate ions and produce proteins that will chelate and keep it sequestered. An overview of the design is nicely provided by the animated video below.
   
   • pArsR
     The first element of the circuit will be a promoter that can respond to the arsenite as well as arsenate concentrations within the cell. BBa_J33201 is a well characterized biobrick that which provides for a promoter whose transcription activity responds to the concentrations of either of arsenate and arsenite with 1uM being sufficient to induce full expression of the downstream protein. ArsR codes for an autorepressor which gets inhibited by presence of Arsenic ions allowing transcription from the promoter to proceed.
   • T7 RNA pol
     Our planned response circuit is a very long cassette and hence transcription of such a long operon would require a very processive polymerase enzyme. Therefore, instead of directly placing the response cassette under pArs, we have first placed expression of T7 RNA pol under pArs so that upon induction by arsenic presence the circuit will first produce T7 RNA polymerase capable to transcribe the downstream operon processively.
     Biobrick Bba_K145001 provides us with such a required gene.
   • pT7
     As we are using T7 RNA polymerase to transcribe our response cassette genes, it is imperative that the entire response cassette be placed under the control of a strong T7 promoter.
   • Synthetic Phytochelatin
     The first major protein produced from the device will be Synthetic phytochelatin. Phytochelatins are glutathione oligomers produced by an enzymatic cascade in algae and other plants to chelate and thereby detoxify heavy metals. iGEM Repository provides for a synthetic phytochelatin coding biobrick Bba_K1321101 expressible under pT7 which serves the desired purpose. The need of the enzymatic cascade is bypassed as the protein can now directly be produced from the processes of central dogma.
   
4. Bacterial Recovery Unit
   As mentioned previously, our mathematical modelling provided us with the insight that the system we have planned will soon saturate. Therefore, a need for a mechanism that can recover the bacterial system from this saturation and push the equilibrium towards uptake of more arsenic, production of more synPC and chelation of more arsenic was felt. Hence, we researched the literature and came across a protein known as HMT that can come to rescue as required. We therefore decided to incorporate HMT in our design.
   HMT1 (Heavy metal transferase 1) is a vacuolar membrane protein from yeast (S. cerevisiae) that is known to transport Cadmium bound synPC complexes from the cytoplasm to the vacuoles. Its heterologous expression in E. coli causes it to be sorted to the inner membrane of the cell wherein it functions to translocate synPC-heavy metal complex to the periplasmic space as no vacuoles are present in bacteria. Interacton of HMT1 with Arsenic bound PC is not yet studied. Our hypothesis is that HMT1 will also work to translocate synPC-As complexes similar what it does with synPC-Cd complexes. This hints at the possibility that HMT1 expression in our system will reduce the overall free synPC, free As ions and synPC-As complex concentration in the cytoplasm thereby pushing the equilibrium forward towards more uptake and more sequestration due to Le Chatelier’s principle. Our mathematical model confirms the same.
   
   Ref: Transport of Metal-binding Peptides by HMT1, a fission yeast ABC type vacuolar membrane protein, Ortiz et. al., JBC 1995
5. A gift in disguise
   The team realizes the limitations of its planned system in successfully sequestering the arsenic in the gut at substantial amounts to make a difference to the suffering patient. It is quite possible that the probiotic bacterial pill might not be able to uptake and sequester large amounts of arsenic from the gut due to saturation. Realizing this, we thought of incorporating in out design a protein that can at least leave the arsenic in the surrounding gut in a less docile form if not completely remove it by sequestration.
   Arsenite oxidase (aox) is an outer membrane protein found in several B-Proteobacterial species that oxidizes the ions in the surrounding media from As(III) form to As(V) form. It is well known that in humans As(III) is highly toxic as compared to As(V). Also, the rate of absorption at the GI epithelium is also greater for As(III) than As(V). Therefore, the greater responsibility of causing arsenicosis is on As(III) than on As(V). Hence, presence of outer membrane protein aox in our system will help to reduce the net concentration of As(III) in the gut by converting it to less toxic As(V) form.
   
   Ref: Arsenite Oxidase (aox) genes from metal resistant B-Proteobacterium, Muller et. al., Journal of Bacteriology, 2003
   
   Note: HMT1 and aox will be in the same operon as synPC all being expressed undet pT7. HMT1 and aox will be IISER Kolkata’s new contributions to the repository.