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What are antibiotics?

Antibiotics, also known as antimicrobial agents are drugs that fight infections caused by bacteria. The key word in the previous sentence is bacteria. Antibiotics will have no effect if taken during a a viral infection like common cold, most sore throats and the flu. However, bacterial infections are a threat too, and antibiotics have served us well in fighting them for over 70 years. So how do they work? Antibiotics are chemicals that interact adversely with different components of a bacterium's structure and/or metabolism, thus bringing about their demise. For example some antibiotics like penicillins attack the cell wall and rupture it, others like tetracyclines target protein synthesis. The diagram below shows some of the molecular targets of antibiotics on bacterial cells.

How does resistance develop?

The emergence of antimicrobial resistance is an evolutionary mechanism. The bacterial population consists of several variants for each of it's gene. Essential proteins are being targeted by most of the antibiotics, which is mostly docking of drug to the protein. Among the variants, few proteins could have a property like they can function but the drug can not dock to the protein anymore. Now, the bacterial cells which have these proteins become resistant.

Antimicrobial peptides

Antimicrobial peptides (AMPs) function differently from the antibiotic molecules as they do not inhibit any protein of the pathogenic bacteria. There are various type of AMPs. In most of the cases, AMP molecules bind to the cell-wall of the bacteria and pierce through the cell wall and break it.

Evolved Resistance Mechanism against anitmicrobial peptide

It has recently been shown that in vitro exposure of bacteria to slowly increasing cationic AMP concentrations over several hundred generations can result in reversible physiological adaptation and/or spontaneous, inheritable resistance to the peptide used. The most observed and hypothesized resistance mechanisms include :

• Alteration of bacterial cell surface charge density and pattern.
• Release of proteases, which break the peptide bonds in the anitimicrobial peptide.
• Trapping or extrusion of antimicrobial peptides.
• Biofilm formation could diminish the activity of antimicrobial peptides.

How would our system help in tackling the emergence of Antibiotic resistance?

As we all are familiar with the theory of natural selection, which says that the species which have adapted it's environment at best would be survive and selected for further generations. Bacteria is a species which has practically lived for billion years. On the course of time, bacteria has evolved to a level which is best for it's survival because of natural selection. The theory of natural selection also talk about trade-off between genes and their function, which is that if you mutate a gene (which gives a functional protein) you might get additional properties from the protein but you would also loose it's inherited property.

Based on the above theory, if we have designed our project to leverage the power of natural selection to eliminate antibiotic resistance. Our idea is being explained following :

We design a system which senses the population of pathogens and secretes the antimicrobial peptide. When we put the pathogenic bacterium under the selection pressure of antimicrobial peptide, most of the pathogens would die and the remaining pathogens might develope a resistance mechanism and can live happily in that environment. These resistant pathogens might follow one or more above mentioned mechanism to acquire resistance against the antimicrobial peptide, but they would be lesser efficient in comparison to normal pathogenic cells in stress-free environment.e.g. if the pathogen cells have changed their cell surface charge density, they might become lesser efficient in the uptake of food, adhere to surfaces etc. compare to a normal pathogenic bacterial cell in stree-free environment. Or if the pathogen cells have evolved proteases, they will not have any advantage over normal pathogen in stress free environment, hence it will not be selected out. etc.

Sept 17

• First meeting of Team:IIT-Madras for iGEM 2015 in our UG lab.
• Ideation begins.☺

Feb15

• Engaged in talk with the mentors about the project, Dr. Nitish Mahapatra and Dr. Kartik Raman.
• Introducing few changes in the project.

May 18-24

• Project being finalized.
• A sketch of proposed work being ready.

May 25-31

• Inventory of all supplies is to be done.
• Alyteserin-1a was chosen to test our model as the structural feature, mechanism of action and other relevent details of anit-microbial peptide Alyteserin-1c, which has two mutations (D4E, N23S), were available in the literature.
• The pdb structure of Alyteserin-1a was generated in pymol, while introducing two mutations D4E and S23N in the pdb structure of Alyteserin-1c.
• The structural features of Alyteserin-1a was analyzed carefully to design a novel peptide which could interact with it.
• Pymol and Pepstr, an online tool, were used to generate a large number of peptide pdb structures of size 10-18 amino acid.
• A software, ZDOCK, was used to assess the docking parameters of Alyteserin-1a and novel peptide.
• Best peforming peptide was chosen to test it's functionality in molecular dynamic simulation.

June 1-7

• Molecul Dynamic Simulation started.
• Made a catalog of all available materials.
• Lactococcus lactis strains, NZ9000 and MG1363, were collected from Prof. KBR's lab.
• MD Simulations finished the proteins were found to interact favourably.

June 8-14

• Started working on the design of genetic circuit.
• One more MD Simulation was performed with the ionic solution of protein complex. MD Simultions showed that naly interacts favorably with Alyteserin forming a cavity of hydrophobic residues.
• Sender is finalised to be E.Coli DH5α, which would constitutively synthesize the AI-2 signaling molecules.
• Receiver is finalised to be Lactococcus lactis NZ9000, which would sense the AI-2 signaling molecules and would behave in the desired way.

June 15-21

• Sequences were finalised for qr1-5 and HFQ.
• Request to iGEM HQ to order extra parts for LuxS, LUXPQUO, Sigma 54, L. lactis constitutive promoter and HFQ.
• MD simulation job in which both peptide were dis-oriented at intial condition was submitted.
• Prepared SOC stock, stored at 4C for autoclaving the next day.
• LB broth, and LB agar was prepared.
• Use of usp45 secretion tag for the secretion of both peptides Alyteserin-1a and NAly.
• In biobrick BBa_K218006, the stop codon for LuxP and the start codon for LuxQ overlap by one base pair.
• Chemical competent cell preparation and transformation over two days. The first transformed batch showed no colonies.

June 22-28

• We started preparation of fresh competent cells. Instead of SOC media, we used only LB broth throughout.
• Agar Stabs for 5 plasmids arrived. We streaked them on agar plates for plasmid isolation on the next day.
• We checked the transformation efficiency using the transformation efficiency kit provided by iGEM. Transformation failed again.
• First plasmid isolation failed with known errors.

June 29 - July 5

• The sequence of all parts and primers to fix the problem in biobrick BBa_K218006 via site-directed mutagenesis, were designed and ordered to IDT.
• Preparation of ultra-competent cells. Tranformation efficiency was found out to be overwhelming.

Aug 10-16

• Plasmid isolation was performed for transformed colonies and other biobrickes which were ordered from iGEM.
• Received the gBlocks and primers from IDT. This delay took a lot from us. :(

Aug 17-23

• Started cloning the gBlocks from IDT. But failed. No overhangs at the 5' and 3' end of the gBlocks therefore we can not use our two RE site, EcoRI and PstI. A big mistake.
• We wished to use the other two REs, XbaI and SpeI, but they would result in self ligation of plasmid backbone and formation of scar at the insert plus backbokne region. We relied on luck.
• Started site-directed-mutagenesis for BBa_K218006. No success.

Aug 24-30

• SDM was repeated 3 times on various possible parameters. Still no success. We gave up on this.
• 2-3 time cloning experiments were performed. No success.

Sept 1-16

• 3A assembly for Biobrick BBa_K622000 and BBa_K1033225