Team:Northwestern/Project

OVERVIEW

Our main project is to simulate mammalian skin using chitin in E. Coli.

In mammalian skin, mitosis occurs in the basal layer of the epithelial cells and cells travel outwards towards the surface of the skin as they mature. As the cells move further away from the basal layer, they die and their cytoplasm is released and the cells are filled with keratin, thus forming a continuously regenerating protective layer on the outer-most part of epithelial layer; our project is modeled after this:

First, a specific strain of Escherichia Coli is used to generate a thick bacterial biofilm lawn.

Then a fast high-expression signalling pathway initiates production of Chitin in the top-most layer of the lawn.

A slow medium-expression signalling pathway initiates production of a cell lysis expression vector in the top-most layer of the lawn.

With both vectors, the E.Coli cells in the top layer generates chitin then lyses, thus depositing a layer of chitin in the top-most layer of the bacterial film.

Whenever there is damage to the chitin layer, the signalling pathway can be reinitiated to regenerate chitin in the damaged location.

The bacterial lawn is externally controlled to produce chitin only at the top-most layer and these cells then undergo apoptosis to result in the formation of a chitinous layer at the surface of the lawn.

First, a thick bacterial lawn is generated by plating Keio Collection ydgG knock-out mutants in an enriched agar plate. The ydgG protein product plays an integral role in AI-2 transport and the knock-outs exhibit increased motility which ultimately allow for a thick lawn.

First, we extracted Chitin Synthase 3 from the Saccharomyces Cerevisiae genome, which we then combined with a lac-operon expression part.

Then we extracted T4 bacteriophage apoptosis cassette from the parts registry - submitted by the '08 Brown iGEM team - from the 2010 iGEM distribution kit. This cassette was again, assembled with a lac-operon expression part.

The lac-operon expression part consists of a constitutive lac repressor expression part followed by a lac-operon attached to a promoter with a ribosome binding site. By altering the lac repressor expression level as well as the ribosome binding site, two distinct expression parts were created: a fast, high-expression part, and a slow (time-delay) medium-expression part. The fast high-expression part was combined with Chitin Synthase, whereas the slow medium-expression part was combined with the apoptosis cassette.

The Chitin Synthase III gene is extracted from the Saccharomyces Cerevisiae genome. Chitin Synthase III catalyzes the polymerization of chitin.

Apoptosis of cells is achieved by using the bacteriophage lysis cassette built by the Brown '08 iGEM Team. The cassette includes Holin, Endolysin, and Rz Protein genes. These enzymes puncture and degrade the cell membrane which results in lysing of the cell and the release of synthesized chitin.

Once the lawn is established, an IPTG solution is sprayed over the lawn to induce chitin synthesis (fast response) and apoptosis (slow response). IPTG mimics allolactose which binds to the LacI repressor, allowing for a build-up in chitin followed by apoptosis and subsequent release into the extracellular environment. Thus, any abrasions to the chitinous surface can be repaired by spraying IPTG.

Strategy

Obtaining Chitin Synthase 3 Gene

First, the Chitin Synthase 3 (CHS3) gene is subcloned out of Saccharomyces Cerevisiae (Baker’s Yeast) cDNA (complementary DNA – contains no introns, thus can be used in prokaryotes) with biobrick restriction sites (EcoR1, Xbal1 on one end, and Spe1, Pst1 on the other). CHS3 was chosen as it was found to be the major enzyme (knockouts had 80% reduced Chitin) in its family and requires no co-enzyme or activating compounds. Upon running CHS3 through NEB Cutter V2.0, we found a PST1 restriction site which is incompatible with the biobrick standard (EcoR1, Xbal, Spe1, Pst1). To remove this Pst1 site, we insert CHS3 into a biobrick vector plasmid, and performed site-directed mutagenesis (SDM).

pMAL Vector

Research revealed that chitin synthase is a transmembrane enzyme, and in order to simulate similar conditions, we decided to use the pMAL vector by NEB which contains a periplasmic membrane signal sequence. CHS3 is subcloned in to the pMAL-p5x NEB vector by first PCR’ing it out of the biobrick vector plasmid - on which it must undergo SDM - with pMAL restriction sites (Nde1 and EcoR1) and ligated into the pMAL-p5x vector. Then, the CHS3 is again amplified with biobrick sites, but this time, with the periplasmic signal sequence native to the pMAL-p5x plasmid, and inserted into a standard biobrick vector. The entire part is then sequenced and submitted to the registry of standard parts. A bacterial apoptosis cassette made by the Brown iGEM team in ‘08 is taken from the iGEM 2010 distribution kit and colony amplified.

Constitutive Promoters & Lac Operon

3 constitutive promoters and 2 lac-inhibitor/lac-operon combination parts are taken from the registry and ligated in all 6 combinations through 3A Assembly. These combinations express different levels of lacI, thus exhibiting variable inductivity. These 6 constructs are then ligated with a reporter protein - green fluorescent protein - and characterized via an automated fluorescent microplate reader; fluorescence is used to determine the construct inductivity and expression level.

IPTG Activation

The data is used to fit our own theoretical kinetics model that accounts for IPTG diffusion through biofilm, external IPTG concentration, internal IPTG concentration, lac repressor concentration, repressor bound gene (DNA) concentration, unbound gene (DNA) concentration, mRNA concentration, enzyme concentration, substrate concentration, enzyme-substrate complex concentration, and final product concentration, as well as all the rate constants involved. Using the data acquired from the microplate reader, a final semi-empirical kinetics model is generated. Using this model, two different constructs are chosen; both with similar inductivity, but one with and the other without time delay.

Chitin Synthesis Production

The fast expression construct is ligated with Chitin Synthase, while the slow expression construct is ligated with the apoptosis cassette. Both plasmids are then transformed into a chiA and tqsA double knockout strain of K12 Escherichia Coli. ChiA knockouts lack Chitinase, which is an enzyme that catabolizes chitin. tqsA knockouts have a disrupted quorum sensing mechanism that allows them to grow very thick lawns. The mutants are acquired from Keio collection (one from Northwestern, and the other from Yale respectively).

MAGE

The Multiplex Automated Genome Engineering (MAGE) protocol developed by H. H. Wang. et al. is used to acquire double knockouts. The MAGE protocol is an accelerated evolution procedure where oligonucleotides are electroporated into the cells in order to introduce mutations in the genome during replication by binding as lagging strand primers, taking advantage of the lambda-red construct.

Mechanism

The double knockout colonies with both constructs are then sprayed with IPTG, which first induces the fast chitin synthesis construct, causi a buildup of chitin in cells in the top layer of the bacterial lawn. Then the slow apoptosis construct is induced, which then lyses the cells, and releases a layer of chitin in the top layer of the bacterial lawn. This construct is then stained with a chitin-specific rhodamine probe by NEB as well as Baclight by Invitrogen in order to ensure that the cells are producing chitin and lysing. The lawn is then imaged through a fluorescent confocal microscope.