Team:MIT MAHE/Parts



The parts we have used in our project.


Two plasmid or composite Bio-Brick designs have been proposed to bring our therapeutic project to life.

Plasmid 1 is a modified mer operon designed for efficient regulation, transport and enzyme production with the control mechanism for Composite Bio-Brick 2 and a reporter gene (SYFP2) present downstream to it on a pSB3K5 vector. It is primarily responsible for detection of methylmercury (or other mercury compounds) using a homodimer protein (MerR), its transport into the bacterial system and production of mercury (II) reductase enzyme and alkylmercury lyase. The gene responsible for the production of SoxR protein which controls the release of Interleukin-10 (IL-10) in Composite Bio-Brick 2 has been integrated into this Bio-Brick to ensure release of anti-inflammatory cytokines (IL-10) only during methylmercury induced inflammation.

Plasmid 2 is designed for the safe and efficient release of the anti-inflammatory IL-10. It would contain the control (SoxS promoter), transport (HlyA transport system) and production gene for IL-10 (fused with the signal peptide of the transport) with the reporter gene (GFP) present downstream to it.

The two plasmids transformed into our probiotic bacteria are proposed to have therapeutic properties for methylmercury poisoning. The individual Bio-Brick sequences are taken from iGEM registry of standard biological parts with the gibson cloning mechanism proposed for ligation.

We have listed the parts derived from different species separately.

Basic Parts

Table 1: List of Basic Parts and their source organisms.
Parts/Bio-BrickWorking Principle
Escherichia coli K-12
SoxR (K554003)SoxR is a transcription factor, under conditions of oxidative stress (presence of nitric oxide (NO) a pro-inflammatory signal) by reversible one-electron oxidation of its [2Fe-2S] cluster. In the active state, it activates the SoxS promoter by untwisting the promoter DNA by binding to the long spacer DNA.
Double Terminator (B0015)It functions to signal the termination of transcription.
Shigella flexneri
MerR + RBS (K346001)MerR is a homodimer and the regulatory factor of the Mer operon. It acts as an activator in the presence of Hg (II) and a weak repressor in its absence, maintaining its expression at a certain level. It binds to the operator site of mercury inducible promoter PmerT. The Hg- bound MerR dimer causes a structural distortion of PmerT, which allows the RNA polymerase to bind, leading to the transcription of genes downstream to it.
PmerT promoter (K346002)PmerT is the mercury inducible promoter of Mer operon. The threshold of PmerT depends on the expression level of MerR.
Serratia marcescens
MerT (K1420005)MerT is a transmembrane protein which receives mercury from MerP at its first transmembrane helix and transports it into the cytoplasm of the bacterial cell.
MerP (K1420003)MerP is the periplasmic component of the Mer transport system which helps in the uptake of mercury inside the cell. It binds to a single Hg (II) ion using its two conserved cysteine residues, which define its metal-binding motif. It detaches attached ligands before passing the Hg (II) on to MerT transmembrane protein.
MerA (K1420001)MerA encodes the mercury reductase enzyme. It reduces Hg (II) to relatively inert and volatile Hg (0) in an NADPH dependent reaction.
MerB (K1420002)MerB encodes the organomercurial lyase enzyme and is usually found immediately downstream to MerA. It catalyses breaking the bond between carbon and mercury through the protonolysis of compounds such as methylmercury.
Aequoria victoria
Bright Yellow Fluorescent Protein SYFP2 (K864100)SYFP2 is a GFP based monomeric protein with optimized folding, maturation, and a narrow fluorescence emission spectrum with a maximum of 515 nm.
GFP/ Green Fluorescent Protein (E0040)Green Fluorescent Protein (GFP) acts as a critical reporter gene to assess the functioning of plasmid by emitting green fluorescence when excited by light in the blue-UV spectrum. Typically, the excitation wavelength measurement range is 430-520 (nm) and the emission wavelength measurement range is 490-580 (nm).
Pseudomonas nitroreducens
SoxS promoter (K554000)SoxS is a conditional promoter, regulated by the SoxR transcription factor. In the active state, SoxR activates the SoxS promoter and starts the transcription of the genes downstream to it.
Escherichia coli O157 H7 strain EDL 933
HlyB/ Hemolysin B (K554007)HlyB acts as an ATP-binding cassette and recognizes the substrate via its secretion signal peptide (HlyA). It exports proteins (like IL-10) fused to the secretion signal produced inside the bacteria that must act on targets outside and is responsible for the specificity of the secretion system process.
HlyD/ Hemolysin D (K554008)It acts as a membrane fusion or adaptor protein by linking the HlyB and TolC domains of the Hemolysin transport system and helps with the transportation of proteins.
TolC (K554009)TolC is a specific outer membrane protein which forms a long channel throughout the periplasm and the outer membrane.
HlyA/ Hemolysin A (K554002)HlyA is a signal sequence that interacts with the cytoplasmic region of the HlyB–D complex. After the binding of the HlyA secretion signal by the HlyB–D complex, HlyD induces the interaction with TolC which then transports it out of the cell.
Homo sapiens (Th1 cell)
IL-10/ Interleukin 10 (K554004)Interleukin 10 (IL-10) is an anti-inflammatory cytokine which plays a role in limiting the host immune response so that the host does not harm its own cells and maintains normal tissue homeostasis.
Parts without a natural comparator
Constitutive Promoter (J23100)Constitutive promoter helps tune the expression levels of constitutively (continuously) expressed parts.
MerC + RBS (K346088)MerC is a transmembrane component of the Mer transport system which helps in the uptake of mercury inside the cell.
RBS with MerE (K1471002)MerE is a transmembrane component of the Mer transport system which helps in the uptake of mercury inside the cell and transport of organomercury compounds.
RBS (B0034)A ribosomal binding site, or ribosome binding site (RBS), is a short sequence of nucleotides present upstream of the start codon of a gene in mRNA.
His-Tag (K1223006)His-tag functions as a purification tag having high affinity to nickel(II) and therefore can be used in affinity purification columns with immobilized nickel ions.

Composite Parts

Table 2: List of Composite Parts designed for experimentation.
Part NumberPart Name
BBa_K3470004Modified mer operon
BBa_K3470005Methylmercury transport system design-1
BBa_K3470006Methylmercury control system
BBa_K3470007Methylmercury Transport system design-1 (Without MerR)
BBa_K3470008Methylmercury Transport system design-2 (Without MerR)
BBa_K3470009Methylmercury Transport system design-3 (Without MerR)
BBa_K3470010Methylmercury Transport system-merP (Without MerR)
BBa_K3470011Methylmercury Transport system-merT (Without MerR)
BBa_K3470012Methylmercury Transport system-merC (Without MerR)
BBa_K3470013Anti-inflammation control mechanism
BBa_K3470014Anti-inflammation transport system
BBa_K3470015Anti-inflammation control mechanism (without SoxR)
BBa_K3470016Methylmercury breakdown mechanism (Without MerA)
BBa_K3470017Methylmercury breakdown mechanism (Without MerB)
BBa_K3470021Methylmercury Transport system-merE (Without MerR)
BBa_K3470022MerA + His-tag
BBa_K3470023MerB + His-tag
BBa_K3470024MerA + MerB + His-tag


Manipal Institute of Technology, Manipal

Manipal Academy of Higher Education

Eashwar Nagar, Manipal, Udupi, Karnataka, India