Team:Westminster/Description

Bed Bugs

Bed bugs have had a long intertwined history with the human world causing a lot of nuisance and disturbance at our homes. Historically it is believed that during the 1940s they were eradicated in the developed world, but in 1995 there has been an increase in their prevalence.

Bed bugs are scientifically called Cimex lectularius and are parasitic on human beings. In general the entire organisms from the Cimicidae family are generally parasitic and specialize to various other organisms. They feed on the blood of warm blooded animals also termed as hematophagy. Cimex lectularius are one such parasitic organism. These are attracted to their host by warmth and CO2 and are very well adapted to the human environment, mostly found in temperate climates.

C. lectularius are small wingless insects that have an oval flattened appearance. They reproduce through traumatic insemination also called hypodermic insemination, where insemination takes place through the body wall, into the body cavity by physical breaching of the epidermis. Males have a hypodermic genitalia that pierces on the abdomen of the female and ejaculates sperm into the body cavity. As these sperm diffuses inside the cavity they reach the ovaries and results in fertilisation. The females can lay 5 eggs in a day and 500 eggs during her lifetime. Eggs are about 1mm long with a milky white tinge and can take 2 weeks to hatch. There are 5 molting stages before they reach maturity.  New-borns are called hatchlings or nymphs and are about the size of poppy seeds. The nymphs start feeding as soon as they hatch and they require one feed in each molting stage. They take about 5 weeks to reach maturity at room temperature environment. Both nymphs and adults are visible from naked eye and there colour might vary from white, light tan to a deep tan or burnt orange. Adults grow about ¼ of an inch long. Female lays 5 eggs in one day and about 500 during her lifetime. Bed bugs can cause adverse health effects to human beings like skin rashes, psychological effects and allergic symptoms.

What is chitin?

Chitin(C8H13O5N)n is a modified polysaccharide which contains nitrogen. It is an abundant polymer found ubiquitously in nature and consists of long chains of N-acetylglucosamine, a derivative of glucose and is similar to cellulose in structure. It forms the main cell wall of many fungi, the exoskeleton of insects and crustaceans (such as crab, lobster and shrimps), the beak and internal shell of cephalopods and the radulas of molluscs. Hydrogen bonding between adjacent polymers gives chitin-polymer matrix increased strength.

In insects, chitin is often modified and embedded in sclerotin (a proteinaceous matrix which forms much of the exoskeleton). This composite material may also be combined with calcium carbonate to produce the much stronger composite of crustacean shells.

Serratia marcescens

Serratia belong to the family Enterobacteriaceae. They are gram negative rod shaped bacteria and are common of many environments from soil, bathroom showers and as a biofilm on teeth.  Isolates are also known to be pathogenic to humans. 

Chitinases from Serratia marcescens

We were kindly gifted the three chitinase genes isolated from S. marcenscens by Prof. Frank Sargent of the Dundee iGEM 2013 team (http://www.lifesci.dundee.ac.uk/people/frank-sargent). In 1969, J.Monreal and E. Reese  found that S. marcescens was the most efficient chitin degrader among 100s of other microorganisms.  We therefore decided to design a chitinase expressing E.coli which would attach out bed bugs.

Mechanism of chitinase activity

There are three main enzymes involved in Chitin degradation in S. marcescens. These have been designated as Chitinase A (ChiA), Chitinase B (ChiB) and Chitinase C (ChiC) Another molecule, CBP21, is also involved in chitin degradation.

ChiA and ChiB primarily bind to chain ends (exo-action). The protein structure of ChiA and ChiB reveals that carbohydrate binding domains are opposite in the 2 enzymes suggesting the two enzymes degrade the polymer from different ends, ChiA from the reducing end and ChiB from the non-reducing end.   Chi C on the other hand is an endo-acting non progressive chitinase which attacks the chitin polymer by making random cuts in the more amorphous regions of the substrate opening these regions for attacks from ChiA and ChiB. ChiA and ChiB are progressive chitinase’s which degrade chitin from opposite ends.

The chitinases cleave the substrate (insoluble polysaccharide) at the α and  β-1,4 bonds in chitin and chito-oligosaccharides producing chitobiose.

REFERENCE

Vaaje-Kolstad, G., Horn, S.J., Sørlie, M., Eijsink, V.G.H., (2013). The chitinolytic machinery of Serratia marcescens--a model system for enzymatic degradation of recalcitrant polysaccharides. The FEBS Journal. 280 (13), 3028. http://onlinelibrary.wiley.com/doi/10.1111/febs.12181/pdf

Site-directed Mutagenisis

One of the most common methods for removing restrinction sites withing gene sequences is to use site-directed mutagenisis – such as the Stratagen method. The Stratagene method uses a forward primer which is 25-45 bases in length and contains the mutation in the centr of the primer sequence. The reverse primer is the complement of this primer. Primers have a minimum GC content of 40% and end in one or more C’s or G’s.

A high-fidelity long range polymerase is required as the whole plasmid is copied in the reaction. The reaction can be visualised as below.

Site-directed Mutagenisis

One of the most common methods for removing restrinction sites withing gene sequences is to use site-directed mutagenisis – such as the Stratagen method. The Stratagene method uses a forward primer which is 25-45 bases in length and contains the mutation in the centr of the primer sequence. The reverse primer is the complement of this primer. Primers have a minimum GC content of 40% and end in one or more C’s or G’s.

A high-fidelity long range polymerase is required as the whole plasmid is copied in the reaction. The reaction can be visualised as below.

Site-directed mutagenisis using overlapping primers

This protocol consists of a tw-step PCR reaction. Mutagenic primers were designed as for the stratagene method. Additionally, forward and reverse primers contining the biobrick prefix and suffix were designed.

PCR reactions using Pfupolymerase was set up. For PCR- A, primers consisted on the forward/prifix primer and the reverse/mutagenic primer. PCR-B consisted of forward/mutagenic primer and reverse/suffix primer. Thus, PCR-A amplified the part upstreamof the mutation site and PCR-B the downstream part of the mutation site f the gene.

The PCR reaction was then run on a gel and the DNA gel extracted. For the second PCR, 2µl of each PCR was added to a mastermix without primers and PCR amplified for 10 cycles. The DNA templates contain overlapping regions which self-prime allowing for extension of the full length of the gene, creating tempate DNA.

After the 10 cycles, primers for the outer regions are added. This ensures amplification of the full gene.

Mutagenic primers are designed as for the stratagene method, using Primer X http://bioinformatics.org/primerx/.

REFERENCE:

Kanoksilapatham, W., Gonzalez, J.M., Robb, F.T., (2007). Directed- Mutagenesis and Deletion Generated through an Improved Overlapping- Extension PCR Based Procedure. Silpakorn University Science and Technology Journal. 1 (2), 7. http://www.journal.su.ac.th/index.php/sustj/article/viewFile/105/112