Biofilm growth

E.coliand B.subtilis cells were made competent by using Buffer 1 and Buffer 2, with their respective compositions shown in Table 1. Both buffers were made in 1L of Ro water. The cells were grown in 300ml LB (Luria-Bertani) broth and incubate in the shaking incubator at 250 rpm at 37C. The sterile LB broth contained 20g LB power and 1L Ro water. The cell cultures were monitored to achieve an absorbance of 0.4- 0.55 nm, cooled on an ice bath for 15 minutes and centrifuged for 15 minutes at 3000 rpm. The supernatants were removed and 50ml of Buffer 1 were added to the tubes and resuspended. They were filled with 24ml Buffer 2 and chilled on ice for 15 minutes. The E.coli and B.subtilis cells have been made competent and ready for transformation (Openwetware.org 2019).

Buffer 1 Buffer 2 Substance Concentration Substance Concentration

RbCl 100mM MOPS (3-(N10mM morpholino) propanesulphonic acid MnCl 2 4H 2 O 50mM RbCl 10mM K acetate 35mM CaCl 2 2H 2 O 75mM CaCl 2 2H 2 O 10mM glycerol 15% w/v glycerol 15% w/v

Table 1. Composition of Buffer 1 and Buffer 2

Transformation by heat shock method

2L of the silk producing spider genes in the vectors pUC19 and pBluescript II SK(-) were added to 50L competent E.coliand B.subtilis using the heat shock method, by chilling the cells in an ice bath, incubating them at 42C for 45 seconds and chilling for 30 minutes. The plasmids were mixed with 450L LB broth and incubated at 37C for one hour. Each mixture was put in 10ml LB broth and shaken at 250 rpm at 37C overnight to grow. Competent E.coliand B.subtilis in water represented the positive control and the plasmids in water the negative control (Biolabs.com 2018).

Blue/white screening of colonies

Blue/white colour selection was performed on using the cultures in order to confirm the successful transformation of the vector plasmid as white colonies. The bacteria with the gene insertion were mixed with LB Broth, incubated at 37C and shaken at 250 rpm. Afterwards, 100L dilutions of the transformants were spread on the LB agar selection plates containing 100mM IPTG, 30mg/ml X-gal, 200mg/ml Ampicillin and Tetracycline, water being used as negative control and bacteria as positive control. The LB agar plates were made of 10g LB powder, 7.5g nutrient agar and 500ml H2O, autoclaved and cooled down to 55C. The performed dilutions were 10 -1 , 10 -2 and 10 -3 (Genscript.com 2018).

Biofilms growth

Successfully transformed colonies were picked using sterile tips, diluted and mixed with 10ml LB broth in 50ml Falcon tubes and grown overnight. Afterwards, 1 ml of each mixture was placed on titanium placed on Petri dishes or in Falcon tubes containing LB Broth and 24-well microtiter plates. Titanium pieces were placed on