silk

Production of silk by biofilms of Bacillus subtilis and Escherichia coli with an insertion of a silk producing gene, grown on different surfaces and validated using IR analysis By

Teodora Popovici 326BMS Final Year Project BSc Analytical Chemistry and Forensic Science March 2019

Page 1 of 24

Contents • •

•

•

• • • • •

Abstract…………………………………………………………...……………3 Introduction……………………………………………………...…………….3 Figure 1. Silk producing gene from the spider Parasteatoda tepidariorum…………………………………………………………………….4 Figure 2. Silk producing gene sequence……………………………….……4 Figure 3. Restriction map of pUC19…………………………………….…...5 Figure 4. Restriction map of pBluescript II SK(-)……………………….……6 Methods………………………………………………………………….…….7 Reconstitution of the silk producing gene and competent cells creation..7 Table 1. Composition of Buffer 1 and Buffer 2……………………………..8 Transformation by Heat shock method……………………………………..8 Blue/white screening of colonies…………………………………………….8 Biofilm growth………………………………………………………………….8 Validation of the biofilms by microtiter assay………………………………9 Silk analysis using Attenuated Total Reflectance technique……………..9 Results…………………………………………………………………………9 Figure 5. Blue/white colonies selection of E.coli …………………………..9 Table 2. Number of colonies in the blue/white screening…………………10 Figure 6. Examples of B.subtilis biofilm and E.coli biofilm formation in 24-well microtiter plates……………………………………………………………......11 Figure 7. Statistical data presenting absorbance (595 nm) of E.coli biofilms in serial dilutions at different incubation times…………………………………12 Figure 8. Statistical data presenting absorbance (595 nm) of B.subtilis biofilms in serial dilutions at different incubation times………………………………12 Figure 9. Comparison of absorbance readings (595 nm) of undiluted E.coli and B.subtilis biofilms at different times of incubation……………………...13 Figure 10. Absorbance (595 nm) of E.coli with inserts in serial dilutions....14 Figure 11. Example of titanium chip submerged in LB broth and host bacteria with insert…………………………………………………………………………14 Table 3. Absorbance (595 nm) readings of LB broth in which biofilms were grown on titanium chips before and after washing………………………......15 Figure 12. IR spectra of natural silk……………………………………………15 Figure 13. IR spectra od synthetic silk………………………………………...15 Table 4. Functional groups and their corresponding wavenumbers……….16 Discussion……………………………………………………………………….16 Table 5. Competent cells efficiency compared to standard efficiency Values……………………………………………………………………………..18 Conclusion……………………………………………………………………….20 Word count………………………………………………………………………20 List of references……………………………………………………………….21 Acknowledgements ……………………………………………………………24

Page 2 of 24

Abstract Silk based compounds play an important role in regenerative medicine, especially in tissue engineering. This experiment aimed to create E.coli and B.subtilis biofilms, which produce silk due to the silk producing gene inserted in the bacterial hosts. The producing silk is verified by IR analysis. E.coli and B.subtilis competent cells were prepared for transformation by heat shock method. A silk producing gene (2ď ­L) carried by two cloning vectors pUC19 and pBluescript II SK(-) was inserted into the bacterial hosts and the recombinant cells were selected by the blue/white colonies selection method. Diluted and undiluted E.coli and B.subtilis biofilms without the insert were grown in 96-well and 24-well microtiter plates at different incubation times in order to determine the best conditions for biofilm creation and biofilms containing the insert were grown in microtiter plates and on titanium chips for 4 hours. The biofilms were validated by microtiter plate biofilm assay and the absorbance (595 nm) of each well was read using a plate reader. The adhesion of the cells on titanium chips was verify by reading the absorbance of the medium in which the titanium was submerged before and after washing and the silk was analysed by ATR, a FT-IR sampling technique. E.coli showed 190 white colonies of recombinant cells and one blue colony on the selection plates (99.4764% recombinant percentage), whilst B.subtilis failed to grow any colony. Moreover, E.coli cells tend to form better bacterial communities than B.subtilis cells without the insert and the best incubation time for both bacteria is 4h. The cloning vector pUC19 presented a higher tendency (up to 2nm absorbance) to create biofilms in the microtiter plates, rather than pBluescript II SK(-) and no biofilms or adhered cells could be determined on the titanium chips. The created biofilms did not produce silk, however synthetic and natural silk samples were analysed by ATR, providing poor, hardly interpretable spectra. It is concluded that silk could not be produced by E.coli and B.subtilis biofilms, however the experiment can be used as guidance for further investigations.

Introduction Parasteatoda tepidariorum is a cobweb spider native to the South-American tropics, however the species has spread worldwide. due to its high reproductive rate, it has come to represent a great interest for developmental and evolutional biology and genetics (Mittmann and Wolff 2012).

Spiders produce different types of silk through the ampullate gland for survival.

However, each type presents a distinct functionality due to its composition. The combination of a dry protein and a wet glue, coated with a wet cement lead to the adhesion property of silk. The mechanical characteristics of silk are given by the total silk proteins or fibroin, which represent large molecules (>250 kDa) composed of long repetitive regions of amino-acids and non-repetitive terminal regions, forming anti-parallel ď ˘-sheets by hydrogen bonds and hydrophobic interactions. (Chaw et al.

2017).

Due to its structural and biochemical properties, synthetic silk and silk based compounds have become a prime focus for regenerative medicine researchers.

Having a many number of applications in tissue engineering, synthetic silk helps

Page 3 of 24

improving the health quality of patients that suffer from cartilage, bone and ligament complications (Das et al. 2014).

The silk producing gene from the spider Parasteatoda tepidariorum represents a circular DNA plasmid and it is illustrated in Figure 1.

Figure 1. Silk producing gene from the spider Parasteatoda tepidariorum (Genscript.com 2019)

The sequence of the silk producing gene with the stop codons being removed so it can be inserted in specific vectors is pictured in Figure 2.

Figure 2. Silk producing gene sequence (Genscript.com 2019) Page 4 of 24

As it can be observed in Figure 1, this plasmid can be cut with five different restriction enzymes (Nhe I, Afi II, Hind III, Kpn I and BamH I) and manipulated for research studies as well as medical uses. The stop codons must be deleted in order to insert the gene into specific vectors, required by the host of interest (Genscript.com 2019). pUC19 and pBluescript II SK(-) are two cloning vectors used for bacterial expression and their restriction maps are presented in Figure 3 and Figure 4 .

Figure 3. Restriction map of pUC19 (Addgene.com 2019)

Page 5 of 24

f1 Ori

T7 promoter

MC

Am p R

S

pBluescript ll SK(-) 3.0kb

T3 promoter lac O lac promoter

Kpn l Xho l Sal l Hind lll EcoR V EcoR l Pst l Sma l BamH l Spe l Xba l Not l Sac l

pUC Ori

Figure 4. Restriction map of pBluescript II SK(-) (Genscript.com 2019)

pUC19 is a small, high-copy number cloning vector with a total length of 2686 bp (basepairs), an origin of replication and a multiple cloning site (MCS) (Coventry University 2017). pBluescript II SK(-) is a phagemid cloning vector with a length of 2961 bp, having polylinker T7 and T3 RNA polymerase promoters present in the Nterminal area of a lacZ gene fragment (Chem-agilent 2008).

Bactria living in communities are scientifically termed ‘biofilms’, which represent a growing interest to microbiologists due to their sophisticated structures and cell-cell communication within the community (Mielich-Süss 2014). Adhesion to a surface represents a critical characteristic for biofilms and it depends on three indispensable components: surfaces, bacteria and an exo-polysaccharide matrix. Firstly, bacterial cells accumulate on the surface, forming a colony under the adsorption process, then secondly the bacterial organisms attach to the surface by creating a polymer layer in between. Finally, the growth and extending of bacteria on the surface.

Biofilm adhesion requires specific medium, pH or temperature for a strong bacterial community creation (Garrett 2008).

Escherichia coli is Gram-negative bacteria, which lives in the environment, food and gastrointestinal tract and it represents the main cause of urinary infections and acute enteritis in humans and animals (Percival et al. 2014). E.coli was established to be a model organism for biofilm creation due to its ideal lifestyle as a community.

Additionally E.coli is an active bacteria; the flagellar apparatus helps for biofilm adhesion and the exo-polysaccharides are necessary for biofilm matrixes (Beloin et al. 2008).

Bacillus subtilis is a Gram-positive bacterium, which grows an animals’ gastrointestinal tract and presents considerable genome diversity. B.subtilis lives in bacterial communities and allows them to manifest complex functions and great interactions among cells, which leads to a durable biofilm (Mielich-Süss 2014).

Page 6 of 24

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 lacZ operon 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 lacZ operon, 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).

Page 7 of 24

E.coli and 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 0.40.55 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).

Substance RbCl

Buffer 1 Concentration 100mM

Buffer 2 Substance Concentration MOPS (3-(N10mM morpholino) propanesulphonic acid RbCl 10mM CaCl22H2O 75mM glycerol 15% w/v

MnCl24H2O 50mM K acetate 35mM CaCl22H2O 10mM 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.coli and 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.coli and 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, 1ml 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 Page 8 of 24

Petri dishes and duplicates containing SGG medium (50ml skimmed milk, 7g glucose and 5ml glycerol) were plated. The biofilms were let to grow for four hours at 37C. In parallel, E.coli and B.subtilis biofilms were grown on microtiter flat and round bottomed plates in different concentrations at 37C for 4h, 24h, 48h and 96h.

Additionally, 96- and 24-well plates were used. Untransformed E.coli and B.subtils cells were used as positive control biofilms and Ro water was used as negative control.

Validation of the biofilms by microtiter assay In order to validate the biofilms, the microtiter assay was performed on each microtiter plate. The medium was discarded by reverse pipetting, using sterile tips and water was added to each individual well. Afterwards, the water was discarded and the plates were incubated in a fume cupboard to dry. When completely dried, the wells were stained with 135L of 0.1% crystal violet solution and incubated at the room temperature for 30 minutes. The crystal violet solution was washed with water by pipetting and the plates were let to dry again. After complete drying, 125L of 30% acetic acid were added to each well and the plates were analysed using a Thermofisher plate reader. The solutions in the 24-well plates were transferred into 96-well flat bottom plates in order to be read. All the plates were read at 595 nm (O’Toole 2011). A microtiter plate containing 30% acetic acid was used to blank the instrument as a negative control. Microtiter plates containing E.coli and B.subtilis biofilms were used as positive controls.

Validation of the biofilms adhesion on titanium The adhesion of the biofilms on the titanium chips was analysed photometically by comparing the absorbance of LB broth in which titanium chips were submerged before and after four washes. The absorbance was read using a Thermofisher spectrophotometer at 595 nm and water was used to blank the spectrophotometer.

Silk analysis using Attenuated Total Reflectance technique Threads of synthetic silk provided from Amazon were partly solubilised by submerging in a calcium chloride:ethanol:water solution in 1:2:8 molar ratio at 37C overnight and natural silk from a common house spider was dissolved in 10% sodium hydroxide at 37C overnight. All the silk samples were let to dry overnight in the fume cupboard. After complete drying, all the silk samples were analysed with Thermofisher FT-IR (Fourrier Transform Infrared Reflectance) apparatus using the ATR (Attenuated Total Reflectance) mode by placing a small amount of the sample on the ATR diamond and run the instrument. Samples of undissolved silk threads were analysed in the same fashion (Coventry University 2018). The obtained spectra were compared to the instrument’s IR (Infrared) spectra library. Atmospheric air as a sample free run was used to blank the instrument and also as a negative control.

Results The experiment was carried out as described in the methods section. Colonies of successfully transformed cells were observed in the blue/white colour selection for recombinants and non-recombinants. White colonies represent the successfully Page 9 of 24

transformed plasmids and the blue colonies are the negative control. They can be observed in Figure 5 below.

Figure 5. Blue/white colonies selection of E.coli Table 2 presents a summary the number of colonies in the agar plates containing the host bacteria and the plasmid vector insertion, undiluted or diluted 10 -1, 10-2 and 10-3.

Plate no.

Bacteria

1

E.coli

Vector

Dilutions

White colonies 66

pBluescript Undiluted II SK(-) 2 E.coli pBluescript 10-1 0 II SK(-) 3 E.coli pUC19 Undiluted 118 -1 4 E.coli pUC19 10 6 5 E.coli - (H2O) Undiluted 0 6 B.subtilis pBluescript Undiluted 0 II SK(-) 7 B.subtilis pBluescript 10-1 0 II SK(-) 8 Control 0 Table 2. Number of colonies in the blue/white screening

Blue colonies 0 0 0 0 1 0 0 0

A free cell agar plate was used as a negative control, which did not show any colony growth. Two plates containing water and the plasmid vectors were also used as negative controls yet did not produce colonies either. Competent E.coli and B.subtilis without the insert were plated as a positive control. As previously observed, E.coli without any insert produced a blue colony, whilst competent E.coli with insertions of plasmid vectors pBluescript II SK(-) and pUC19 produced white colonies as successful recombinant plasmids. Undiluted E.coli with the vector pBluescript II SK(-) produced 66 white colonies and the viable competent cells can be calculated as follows 66(colonies) x 10ml= 660 = 6.60 x 102 cells/ml. On the plate containing Page 10 of 24

undiluted E.coli with pUC19, 118 colonies appeared colonies, leading to the calculation of the viable competent cells 118(colonies) x 10ml= 1180 = 1.118 x 103 cells/ml. One dilution of E.coli, which took up the plasmid vector pUC19, presented 6 colonies and the viable competent cells are calculated as follows 6(colonies) x 10ml x 10 (dilution factor) = 600 = 6 x 102 cells/ml. The recombinant percentage of the selection was calculated as follows: recombinant percentage=white colonies x 100/ white +blue colonies= 190 x 100/ 191 = 99.4764%. The plates containing the rest of the dilutions did not show any colonies.

Serial dilutions of E.coli and B.subtilis biofilms, containing the competent cells and LB broth were produced in 96-well and 24-well microtiter plates and incubated for different periods of time. 100ď ­L volume of mixture was used for the 96-well plate and 1ml and 2ml volume for the 24-well plate. Biofilms were observed on the bottom of the wells, however 2ml volume of solution in the 24-well plate presented circular biofilms on the side of the well. Figure 6 illustrates examples of B.subtilis and E.coli biofilms on the bottom of well and on the side of the well in a 24-well microtiter plate.

The working volume used was 2ml and the plate has been stained with 0.1% crystal violet solution and dried naturally.

Figure 6. Examples of B.subtilis biofilm (left) and E.coli biofilm (right) formation in 24-well microtiter plates The competent E.coli and B.subtilis biofilms were incubated for 4 hours, 8 hours, 24 hours, 48 hours and 96 hours in order to determine the most beneficial conditions for biofilm creation. The biofilm solutions were produced by serial dilution technique (undiluted, 1 in 10 and 1 in 102) and the absorbance of each well and each microtiter plate was read using a plate reader at 595 nm. Figure 7 and Figure 8 show the absorbance readings at different incubation times for diluted and undiluted E.coli and respectively B.subtilis without the silk gene insert biofilms.

Page 11 of 24

Figure 7. Statistical data presenting absorbance (595 nm) of E.coli biofilms in serial dilutions at different incubation times

Figure 8. Statistical data presenting absorbance (595 nm) of B.subtilis biofilms in serial dilutions at different incubation times Page 12 of 24

According to Figure 7, undiluted E.coli biofilms tend to have relatively steady absorbance values, 4h and 96h incubations show absorbance of approximatively 1 nm. Samples diluted 1:10 indicate the highest absorbance at 96h incubation time with a difference of 0.5 nm between the absorbance of the 8h incubated samples.

The samples diluted 1:100 present imbalanced absorbance values, the 96h incubation time representing the highest value.

Regarding Figure 8, the absorbance of undiluted B.subtilis biofilms vary significantly, as the 8h incubation showing a negative value and the 4h incubation the highest absorbance. The 1:10 diluted samples appear to have the highest absorbance values, the 8h incubation time being the most favourable one. The 1:100 dilution shows great absorbance at 48h incubation, however 96h incubation time indicates no absorbance.

Figure 9. Comparison of absorbance readings (595 nm) of undiluted E.coli and B.subtilis biofilms at different times of incubation A comparison of E.coli and B.subtilis biofilm growth is illustrated in Figure 9. E.coli biofilms show greater absorbance, the values being situated between 0.8 and 1.1 nm, whilst B.subtilis biofilms present no absorbance value at 8h incubation time and unsteady absorbance readings throughout all incubation times. Overall, 4h incubation time indicates a relatively high absorbance for both biofilms.

Figure 10 depicts the absorbance readings of diluted and undiluted E.coli biofilms with different inserts incubated for 4h.

Page 13 of 24

Figure 10. Absorbance (595 nm) of E.coli with inserts in serial dilutions The undiluted E.coli with pUC19 biofilm shows the highest absorbance reading (3.4 nm), whilst E.coli with pBluescript II SK (-) and without any plasmid show low values of absorbance situated between 0.5 and 1.5 nm. Diluted E.coli with pUC19 biofilm maintain constant values of absorbance throughout the serial dilutions.

E.coli and B.subtilis biofilms were grown on titanium chips, which were submerged in LB broth and kept under observation. An example of submerged titanium is illustrated in Figure 11.

Figure 11. Example of titanium chip submerged in LB broth and host bacteria with insert After 4h incubation, the titanium chips were washed and the absorbance of LB broth before and after the washing was measured at 595 nm. Table 3 depicts the absorbance values of LB broth before and after washing.

Page 14 of 24

Sample

Absorbance (nm) Absorbance (nm) after before wash wash B.subtilis 0.56 0.558 E.coli+pUC19 0.585 0.58 E.coli+pBluescript II KS(-) 0.558 0.553 Table 3. Absorbance (595 nm) readings of LB broth in which biofilms were grown on titanium chips before and after washing As it can be observed, the absorbance values for each sample do not present a significant difference before and after the wash.

Natural silk from a common house spider and synthetic silk were dissolved then analysed by the ATR technique, recording the reflectance of the crystals in the silk.

The produced spectra were compared with IR spectra library from the apparatus in order to determine the composition, by a matching percentage. Figure 12 illustrates spectra of natural silk, whilst Figure 12 shows spectra of synthetic silk.

Figure 12. IR spectra of natural silk As it can be observed, natural silk presents a resolved peak at 3300 cm-1, however the absorption bands between 2300 and 1400 cm-1 are less easily interpretable. The IR library found that Manganese(II) nitrate hydrate presents a composition matching percentage of 50.26% with the sample.

Figure 13. IR spectra of synthetic silk Figure 13 illustrates an IR spectra of dyed synthetic silk and presents a relative similarity with the analysed natural silk spectra. The synthetic silk peaks at 3300, 1600 between 1300 and 900 cm-1. The IR library a composition matching rate of 58.53% with cellophane.

Page 15 of 24

Table 4 represents the chemical functional group corresponding in the IR spectra region, which helps to identify the structure of the compounds. The functional class was chosen for stretching vibrations.

Wave number (cm-1) Functional groups 722 (CH2)n 750-870 C-H in aromatics 1400 N-O asymmetric 1100 n(C-O) 1200 C-H (Amide III) 1650 C=O (Amide II) 2860 CH2 2960 CH3 3200 N-H (Amide I) 3400 OH Table 4. Functional groups and their corresponding wavenumbers (Rani et al.

2017; Ciolacu, Ciolacu and Popa 2010).

Natural and synthetic silk did not reflect properly, resulting in poor IR spectra. The natural silk presented a peak at 1400 cm-1, representing the nitro (NO) functional group and the absorption region between 3400 and 3200 cm-1 the hydroxyl group (OH) was a result of the ethanol used in the dissolving process. The absorbance band situated at around 1000 cm-1 represents the ď ˘-sheets constructing the silk fibroin. The IR library found similarities in silk’s composition with Manganese(II) nitrate hydrate and Trimethylboroxine. On the other hand, the dyed synthetic silk spectra showed unsteady changes in the 3600-2400 cm-1, comparing to the natural silk. The n(C-O) groups appeared around 1100 cm-1 and the hydroxyl class at 3400 cm-1, and moreover, the larger peak around 3400 cm-1 can represent amounts of bound water, which contribute to the cellulose IR spectrum. The IR library found a relatively higher similarity of the synthetic silk composition with cellophane, which is a transparent sheet made of cellulose.

Discussion Competent E.coli and B.subtilis cells were prepared for transformation. E.coli cells presented moderated efficiency transformation, whereas B.subtilis strain suffered total inefficiency in transformation, due to the competent cells preparation. One technique was chosen for both bacterial hosts, however B.subtilis cells led to failure in the blue/white colonies selection step due to the incompatibility of the used competent cells preparation technique. Competent B.subtilis cells preparation is more complex and it requires expression of B.subtilis competence transcription factor ComK from an IPTG-inducible promoter carried by an unstable plasmid to generate the genes required for the DNA uptake and further recombination. The ComK factor will be lost after transformation due to its intrinsic instability and further genetic manipulation can be performed without any interferences (Nijland et al.

2010).

The blue/white colonies screening was performed in order to select the successful recombinant bacterial hosts, which have taken up the silk producing gene. The agar Page 16 of 24

plates containing diluted and undiluted E.coli with both inserts presented 190 white colonies, representing the recombinant cells and one blue colony, indicating the galactosidase presence detected by X-gal. The host E.coli strain contains the lacZ deletion mutant, scientifically termed lacZM15, caring a mutant -galactosidase mutant with deleted 11-41 N-terminal residues, termed the -peptide. The vectors pUC19 and pBluescript II SK(-) carry the lacZ sequence which encodes the first 59 residues of -galactosidase, producing the -peptide. The white colonies show no functional -galactosidase enzyme, whilst the blue colonies indicate hydrolyzed Xgal and no interruption of lacZ (Aitken 2012).

On the other hand, the agar plates containing transformed B.subtilis cells did not show any colonies. Regardless whether both vectors contain the lacZ, B.subtilis does not carry the lacZ operon, leading to an unsuccessful blue/white colonies selection. Glycolic genes cggR, gapA, pgk, tpi, pgm and eno are transcribed as a hexacistronic operon, named gapA, repressed by the CggR regulator (Ludwig et al.

2001). A scientifically proved effective way to induce -galactosidase enzyme activity in B.subtillis is the use of the lacZ gene from E.coli as a reporter gene, where the lacZ sequence is attached to B.subtillis and use the blue/white colonies selection (Daniel et al. 1997). Another way to allow selection in presence of antibiotic in B.subtilis cells is the use of antibiotic-resistance cassettes containing genes encoding resistance to the antibiotics erythromycin, kanamycin, tetracycline and spectinomycin, which were cloned in the polylinker of E.coli plasmid vectors (Guérout-Fleury et al. 1995).

The performed dilutions were 10-1, 10-2 and 10-3 and the resulted colonies could be countable, however, the 10-3 dilution did not show any colony on the plates. More dilutions could have been performed for more detailed investigation and accuracy.

The recombinant percentage was calculated to be 99.47%, showing the efficiency of the technique applied on E.coli cells. However, the viable competent cells were calculated to be 6.60 x 102, 1.118 x 103 and 6 x 102 cells/ml. Table 4 presents the viable competent cells expressed in cfu (colony forming units)/g and a categorization of the competent cells efficiency according to Thermofisher standards (Thermofisher.com 2019).

Page 17 of 24

Bacterial host and vector

Viable competent cells (cells/ml)

Viable competent cells (cfu/g)

High (gold) efficiency >109 cfu/g

Medium (silver) efficiency 108-109 cfu/g

Subcloning (bronze) efficiency 106-107 cfu/g or less ✓

Undiluted 6.60 x 102 6.60 x 105 E.coli+pBluescript II SK (-) Undiluted 1.118 x 1.118 x ✓ E.coli+pUC19 103 106 E.coli +pUC19 6.00 x 102 6.00 x 103 ✓ diluted 1:10 Table 5. Competent cells efficiency compared to standard efficiency values (Thermofisher.com 2019)

Agar plates containing E.coli cells and the insert showed bronze efficiency or even less viable competent cells to be categorized, whilst plates containing B.subtils cells showed total inefficiency of the competent cells.

Biofilms were grown in 96-well and 24-well microtiter plates in different volumes. As it can be observed in Figure 6, B.subtilis biofilms were formed on a 24-well plate well in 2ml volume grew on the bottom of the well, as well as on the side of it. E.coli biofilms were created on the bottom of the well. The 96-well plates indicated presence of E.coli and B.subtilis biofilms on the bottom of the wells, however this can represent a false positive due to the possibility of accumulation of the cells on the bottom of the well (Coventry University 2019).

Serial dilutions of non-recombinant E.coli and B.subtilis incubated at different times were used in creating the efficient conditions for biofilm formation. According to Figures 7 and 8, the biofilms showed a greater absorbance while diluted 1:10 with LB broth, indicating that the cells tend to grow more as bacterial communities in presence of LB broth rather than dry cells. The most beneficial incubation time for E.coli biofilms was determined to be 96h, whilst the best incubation time for B.subtilis biofilms was 4h. Conclusively, the 4h incubation time showed good absorbance values for both E.coli and B.subtilis biofilms. LB broth was used as negative control, however the absorbance readings indicated high values, showing contamination in the wells.

Recombinant E.coli showed a greater potential to create biofilms with the pUC19 insert for both diluted and undiluted samples, whilst the plasmid vector pBluescript II SK (-) inserted within the bacterial host demonstrated a tendency to remain planktonic cells more than E.coli with no insert.

Both pUC and pBluescript variations present a high-copy number, ampicillin resistance and a wide range of restriction endonucleases can be used to cut them for further cloning procedures. However, one difference between the two cloning vectors is that pBluescript vectors carry flanking promoters in the MSC, which allow the insert DNA transcription on either strand. Being phagemids, pBluescript vectors Page 18 of 24

contain a single-stranded filamentous bacteriophage origin of replication, termed M13 phage and therefore single-stranded DNA applications, such as DNA sequencing or site mutagenesis present a higher efficiency rather than replicating conventional double-stranded plasmids (Casali and Preston 2003). Thus E.coli with pBluescript II SK(-) did not produced effective biofilms.

Silk was expected to be produced by the biofilms due to the silk producing gene insert of both E.coli and B.subtilis hosts. The silk gene was first introduced into two different cloning vectors, pUC19 and pBluescript II SK(-) and inserted into the bacterial host to replicate. Cloning vectors are small segments of DNA, used to insert genes into host cells with the purpose of obtaining numerous copies of the gene insert, however they are not able to express the functions of the of the genes of interest. Expression vectors are DNA plasmids, which transcript and translate specific gene inserts into the host cells, forcing the mechanisms of the cell to produce the relevant gene. An expression vector is able to comprise enhancers, strong promoter region, termination codon, transcription and translation initiation sequences, origin of replication, restriction sites and a selectable marker, allowing the silk gene to successfully produce silk fibroins in the biofilms (Lakna 2017).

The microtiter plate biofilm assay was performed to determine whether biofilms where formed or not on the microtiter plates. This technique is based on staining the plate wells with crystal violet solution, then washing the dye with acetic acid solution for a proper preparation for absorbance reading. The results showed that E.coli biofilms indicate greater absorbance values than the B.subtlis biofilms. E.coli is Gram-negative bacteria and B.subtilis is a Gram-positive bacteria, the difference between these two groups is the colour change after staining with crystal violet solution due to the composition of the cell wall. This issue can lead to false positives in the microtiter plate assay. Gram-positive bacteria remain dark violet after staining due to the multi-layered peptidoglycan with embedded proteins cell wall which retain the purple colour of the stain. On the other hand, the cell wall of Gram-negative bacteria contains an extracellular membrane with a periplasmic space layer between the membrane and the peptidoglycan layer and does not allow the stain to be retained, resulting a fade red-pink colour after Gram staining (Thairu et al 2014).

An attempt of biofilm creation on titanium chips led to failure as no sight of bacterial community could be observed on titanium after 4h incubation. A possible cause of the failure could be the inability of the bacteria to adhere to the surface and form a biofilm further on. During bacterial adhesion, planktonic cells swim using the flagella or are attracted to the surface by Brownian motion, van der Walls attraction, gravitational forces, surface electrostatic charge and hydrophobic interaction, leading to irreversible adhesion. Bacterium’s capsule, flagella and pili help the organism to adhere to the surface. Hydrophobicity, chemical composition, roughness and topography of the surface can influence the adhesive behaviour, however surface coatings can bring an important contribution to solving this issue. Polymer coatings can promote cell adhesion, stimulating the exo-polysaccharide matrix formation if chosen correctly. Titanium surface can be coated with polymers by plasm polymerisation or electro-polymerisation. For instance, hexamethyldisiloxane coating could be fasten the attachment of the extracellular matrix, whilst polymer layers functionalised by N-acetyl-GCRGYGRDGSPG peptide can be beneficial for specific protein matrixes. Moreover, pre-treated titanium by grit blasting, acid etching or Page 19 of 24

plasma/laser treatment generate a stronger substrate-coating bonding and thus, firmer biofilm adhesion (Wang 2011).

The natural and synthetic silk were analysed using ATR, providing IR spectra. The composition of both types of silk was meant to be used as standards and compared to the composition of silk produced by the biofilms. However, regardless the failure of silk production, the experiment was carried out with the purpose of helping further investigations.

The presented IR spectra can be categorized as poor analysis due to the shape of the absorbance bands, the interpretation being relatively difficult, yet the IR library provided profiles of compounds with similar composition to identify the samples. An attempt of interpretation was done in order to justify the similarities with the compounds found by the IR library. As the results show, the natural silk’s crystalline domains reflected poorly, providing an IR spectra with wide bands. The intense bands resulted from the amide vibrations, representing the coupling of the peptide bonds in the cell. The spectral regions associated with the amide vibrations are 3200, 1600 and 1200 cm-1 (Rani et al. 2017).

The synthetic silk composition was determined to be cellulose. The broad band situated between 3600 and 3000 cm-1 spectral region resulted from the OH stretching vibration caused by the hydrogen bonds. The 2900 cm-1 band correspond to the C-H stretching vibration, determining the composition of cellulose (Ciolacu, Ciolacu and Popa 2010). A better characterization of silk could be given by scanning electron microscopy analysis, where elucidation of the complete sequence of silk proteins is possible, as well as a 3D structure (dos Santos-Pinto et al 2018).

Conclusion The aim of the experiment was to create and investigate strong E.coli and B.subtilis biofilms, which can produce silk due to the silk producing gene inserted into the bacterial hosts. Unfortunately, this aim was not reached due to the wrong choice of the vectors in which the silk gene was ordered, wrong competent B.subtilis cells production and inability of the cells to attach to the titanium chips in order to form a biofilm. The information provided by this experiment could be used for further investigations.

Word count: • Abstract: 336 words • Introduction: 998 words • Methods: 943 words • Results: 1343 words • Discussion: 1697 words • Conclusion: 85 words • TOTAL: 5402 words

Page 20 of 24

List of references

Addgene.org. (2019). Addgene: Vector Database - pBluescript II SK (-). [online] available from https://www.addgene.org/vector-database/1947/ [ 24 Mar. 2019].

Addgene.org. (2019). Addgene: Vector Database - pBluescript II SK (-). [online] available from https://www.addgene.org/vector-database/1947/ [24 Mar. 2019].

Casali N. and Preston A. (2003). E. Coli plasmid vectors: Methods and applications, Vol-235. Totowa: Humana Press.

Ciolacu, D., Ciolacu, F. and Popa, V. (2010). Amorphous cellulose- structure and characterization. Cellulose chemistry and technology, 45, pp.13-21.

Coventry University (2017). 226BMS Biomolcular Analysis LABORATORY CLASSES 2017-2018 DNA cloning Labs 1-3 Coventry: Coventry University Coventry University (2017). 226BMS-Biomolecular Analysis Coventry: Coventry University Daniel, R., Haiech, J., Denizot, F. and Errington, J. (1997). Isolation and characterization of the lacA gene encoding beta-galactosidase in Bacillus subtilis and a regulator gene, lacR. Journal of Bacteriology, 179(17), pp.5636-5638. dos Santos-Pinto, J., Arcuri, H., Esteves, F., Palma, M. and Lubec, G. (2018). Spider silk proteome provides insight into the structural characterization of Nephila clavipes flagelliform spidroin. Scientific Reports, 8(1).

Garrett, T., Bhakoo, M. and Zhang, Z. (2008). Bacterial adhesion and biofilms on surfaces. Progress in Natural Science, 18(9), pp.1049-1056.

GuĂŠrout-Fleury, A., Shazand, K., Frandsen, N. and Stragier, P. (1995). Antibioticresistance cassettes for Bacillus subtilis. Gene, 167(1-2), pp.335-336.

Kasoju, N. and Bora, U. (2012). Silk Fibroin in Tissue Engineering. Advanced Healthcare Materials, 1(4), pp.393-412.

Lakna (2017). Difference Between Cloning Vector and Expression Vector | Definition, Types, Uses, Similarities, Differences. [online] Pediaa.Com. available from http://pediaa.com/difference-between-cloning-vector-and-expression-vector/ [20 Mar. 2019].

Ludwig, H., Homuth, G., Schmalisch, M., Dyka, F., Hecker, M. and StĂźlke, J. (2001).

Transcription of glycolytic genes and operons in Bacillus subtilis: evidence for the presence of multiple levels of control of the gapA operon. Molecular Microbiology, 41(2), pp.409-422.

McKeen, L. (2012). Film properties of plastics and elastomers. [Place of publication not identified]: William Andrew.

Page 21 of 24

Mittmann, B. and Wolff, C. (2012). Embryonic development and staging of the cobweb spider Parasteatoda tepidariorum C. L. Koch, 1841 (syn.: Achaearanea tepidariorum; Araneomorphae; Theridiidae). Development Genes and Evolution, 222(4), pp.189-216.

Nhm.ac.uk. (2019). [online] available from http://www.nhm.ac.uk/content/dam/nhmwww/our-science/dpts-facilitiesstaff/Coreresearchlabs/blue-white-colony-screening_aug12-118543.pdf [ 24 Mar. 2019].

Nijland, R., Burgess, J., Errington, J. and Veening, J. (2010). Transformation of Environmental Bacillus subtilis Isolates by Transiently Inducing Genetic Competence. PLoS ONE, 5(3), p.e9724.

O'Toole, G. (2011). Microtiter Dish Biofilm Formation Assay. Journal of Visualized Experiments, (47).

Openwetware.org. (2019). Wiese Lab:Competent Cell Prep - OpenWetWare. [online] available from https://openwetware.org/wiki/Wiese_Lab:Competent_Cell_Prep [ 21 Mar. 2019].

Percival, S., Yates, M., Williams, D., Chalmers, R. and Gray, N. (2014). Microbiology of waterborne diseases. Elsevier.

Pumure, I., Ford, S., Shannon, J., Kohen, C., Mulcahy, A., Frank, K., Sisco, S. and Chaukura, N. (2015). Analysis of ATR-FTIR Absorption-Reflection Data from 13 Polymeric Fabric Materials Using Chemometrics. American Journal of Analytical Chemistry, 06(04), pp.305-312.

Rani, K., Chandwani, N., Kikani, P., Nema, S., Sarma, A. and Sarma, B. (2017).

Optimization and surface modification of silk fabric using DBD air plasma for improving wicking properties. The Journal of The Textile Institute, 109(3), pp.368375.

Sangunamani, D., Raghu, K. and Naik, S. (2019). ATR-FTIR Spectroscopy analysis of Silk. [online] available from https://www.chemarc.com/content/article/atrftirspectroscopy-analysis-of-silk/5c4a9afa6b17990f29c82d6d [ 21 Mar. 2019].

Siesler, H. (1980). Fourier transform infrared (ftir) spectroscopy in polymer research. Journal of Molecular Structure, 59, pp.15-37.

Thairu, Y., Usman, Y. and Nasir, I. (2014). Laboratory perspective of gram staining and its significance in investigations of infectious diseases. Sub-Saharan African Journal of Medicine, 1(4), p.168.

Thermofisher.com. (2019). Chemically Competent Cells | Thermo Fisher Scientific UK. [online] available from https://www.thermofisher.com/uk/en/home/lifescience/cloning/competent-cells-for-transformation/chemicallycompetent.html#medium [ 19 Mar. 2019].

Page 22 of 24

Van Die, I., Bergmans, H. and Hoekstra, W. (1983). Transformation In Escherichia coli: Studies On The Role Of The Heat Shock In Induction Of Competence. Microbiology, 129(3), pp.663-670.

Wang, A. (2011). Bacterial Biofilms and Biomineralisation on Titanium., Birmingham: University of Birmingham

Page 23 of 24

Acknowledgements

I would like to express my appreciation to Dr. Sharon Williams for her guidance and help during this project. Without her valuable assistance, as well as the encouragement at various stages of the project period, I would not have achieved my best work.

I am also very thankful to the laboratory technicians during the data collection period for all their hard work to satisfy any need for my experiment.

Finally, I would like to thank the Faculty of Health and Life Sciences for offering me all the opportunities to reach my aims with cutting-edge technology and friendly staff that were constantly willing to help.

Teodora Popovici

Page 24 of 24