Reconstitution of the silk producing gene and competent cells creation

In order for bacteria to take up the plasmid, transformation and blue/white colonies selection procedures must be performed. Transformation represents the process through competent bacterial cells are able to uptake by exposure to heat shock in the presence of divalent cations, which counteract the electrostatic repulsion between the bacterial membrane and the plasmid. The drastic change of temperature in the heat shock method support the plasmid insertion into the bacterial host by opening and closing pores into the bacterial membrane (Van Die et al. 1983). Blue/white screening represents an efficient method for recombinant bacteria identification. The presence of lactose triggers the lacZoperon in the host bacteria, producing -galactosidase enzyme, which cleaves lactose into glucose and galactose. When a foreign DNA is taken up by the host, the -galactosidase enzyme is not produced. If the host did not accept the plasmid, -galactosidase enzyme is produced and X-gal (5-bromo-4-chloro-3-indolyl--D-galactopyranoside) is hydrolysed to 5-bromo-4-cloro-indoxyl, developing a blue pigment and therefore nonrecombinant cells appear blue and recombinant ones white. An artificial inducer to the lacZoperon, IPTG (isopropyl -D-1 -thiogalactopyranoside), is used along with Xgal, which stimulates transcription but no enzyme activity (sigmaaldrich.com 2019).

Attenuated total reflectance (ATR) is a Fourier transform infrared (FT-IR) spectroscopy sample technique, that can be used to analyse silk fibroins and it uses an optically dense crystal (diamond) with a high refractive index at a specific angle to measure the internally reflected IR beam, which changes when it comes in contact with the sample. The light beam is conducted onto the diamond, which creates an evanescent wave that travels beyond the crystal surface into the sample, that is kept in contact with the diamond. The evanescent wave is attenuated when the sample absorbs energy and returns to the diamond, exits on the opposite end and is guided to the IR spectrometer, generating IR spectra (Thrmofisher.com 2019).

The experiment was aimed to create and investigate durable biofilms that have a high tensile strength in B. subtilis and E. coli, grown on different surfaces. A silk producing gene was inserted into the hosts so the biofilms create silk used in regenerative medicine development.

Methods

The procedure was approved by Genetic Modification Safety Committee, committee number 45.

Reconstitution of the silk producing gene and competent cells creation

The Genscript silk producing gene extracted from the spider Parasteatoda tepidariorum in the cloning vectors pUC19 and pBluescript II SK(-) were reconstituted according to Genscript.com instructions by adding 20L sterile milli Q water, vortexing for one minute and then cooled in an ice bath (Genscript.com 2019).