Disc Experiment 1

We prepared an experiment like the disc experiments methods used to see antibiotic effectiveness, in order to see the effect of B. Subtillis with LALF on E. Coli.

By adding E. Coli on Bacillus Subtilis;

Plate A1); We put E. Coli on the B. Subtilis with LALF (K541915) biofilm. For his purpose we first prepared a corn starch with liquid LB medium and added 100 ul B. Subtilis liquid medium as a point over it. For a qualified biofilm we incubated it for 24 hours at 37˚C.

Later we added 10 uL E. Coli with RFP as a single point and again incubated it.

Plate A2); In order to understand whether LALF protein or another factor killed E. Coli on the Bacillus biofilm we prepared the same contrivance with a B. subtilis biofilm that doesn’t produce LALF protein or doesn’t have antibiotic resistance. So we could understand if the B. Subtilis biofilm itself not LALF, inhibited the growth of E. Coli.

Plate A3);At this contrivancethe B. Subtilis we used for biofilm didn’t includeLALF protein but had antibiotic resistance. Why we didi this is we knew that there would be a color difference between Plate A1 and A2 but we woluld like to know if the antibiotic would prevent E. Coli with RFP from having color.

Plate B1); We spreaded B. Subtilis with LALF over a normal lb medium. We put E. Coli as a single point while the Bacillus spread is fresh. We aimed to see if B. Subtilis could kill E. Coli without forming a biofilm.

Plate B2); We prepared this plate as a control plate of Plate C. For thşs we prepared the same contrivance with only B Subtilis that doesn’t produce LALF protein or doesn’t have antibiotic resistance in order to be sure that if the EColi of the plate B1 does not grow the reason for this is LALF protein.

Plate B3); In this contrivance we spread B. Subtilis that doesn’t produce LALAF protein but has antibiotic resistance and added E.Coli with RFP as a single drop over it.

By adding E. Coli over B. Subtilis ;

Plate C1); We added 100 ul liquid culture of B. Subtilis with LAFL (k541915), over the normal lg medium applied with E. Coli with RFP,as a single drop.

Plate C2); Over the fresh spread of E. Coli, 100 ul of liquid culture of B. Subtiis not producing LALF or doesn’t have antibiotic resistance as a single drop.

Plate C3); At this contrivance we added B. Subtilis that doesn’t produce LALF but has antibiotic resistance over the fresh spread of E. Coli with RFP likewise. We aimed to see the affect of antibiotic resistance over E. Coli with RFP ,by using B. subtilis with and without antibiotic resistance.

Plate D1); We added supernatant of B. Subtilis with LALF as a single drop over fresh spread of E. Coli with RFP. Because at the beginning of the LALF part of B. Subtilis there was a throwing out signal and we expected death mostly here.

Plate D2); At this plate we applied 100 ul of supernatant of B: subtilis not producing LALF protein and not having antibiotic resistance over the fresh spread of E. Coli as a single drop.

Plate D3); At this plate we applied 100 ul of supernatant of B: subtilis not producing LALF protein and but that has antibiotic resistance over the fresh spread of E. Coli as a single drop.

 

Plates B2 and B1 (Respectively)

Plates C1 and C2 (Respectively)

Plates D2 and D1

DISC EXPERIMENT 2

We prepared an experiment like the disc experiments methods used to see antibiotic effectiveness, in order to see the effect of B. Subtillis with LALF on E. Coli.

By adding E. Coli on Bacillus Subtilis;

Plate A1); We put E. Coli on the B. Subtilis with LALF (K541915) biofilm. For his purpose we first prepared acorn starch with liquid LB medium and added 100 ul B. Subtilis liquid medium as a point over it. For a qualified biofilm we incubated it for 24 hours at 37˚C.

Later we added 10 uL E. Coli with RFP as a single point and again incubated it.

Plate A2); In order to understand whether LALF protein or another factor killed E. Coli on the Bacillus biofilm we prepared the same contrivance with a B. subtilis biofilm that doesn’t produce LALF protein or doesn’t have antibiotic resistance. So we could understand if the B. Subtilis biofilm itself not LALF, inhibited the growth of E. Coli.

Plate A3);At this contrivancethe B. Subtilis we used for biofilm didn’t includeLALF protein but had antibiotic resistance. Why we didi this is we knew that there would be a color difference between Plate A1 and A2 but we woluld like to know if the antibiotic would prevent E. Coli with RFP from having color.

Plate B1); We spreaded B. Subtilis with LALF over a normal lb medium. We put E. Coli as a single point while the Bacillus spread is fresh. We aimed to see if B. Subtilis could kill E. Coli without forming a biofilm.

Plate B2); We prepared this plate as a control plate of Plate C. For thşs we prepared the same contrivance with only B Subtilis that doesn’t produce LALF protein or doesn’t have antibiotic resistance in order to be sure that if the EColi of the plate B1 does not grow the reason for this is LALF protein.

Plate B3); In this contrivance we spread B. Subtilis that doesn’t produce LALAF protein but has antibiotic resistance and added E.Coli with RFP as a single drop over it.

By adding E. Coli over B. Subtilis ;

Plate C1); We added 100 ul liquid culture of B. Subtilis with LAFL (k541915), over the normal lg medium applied with E. Coli with RFP,as a single drop.

Plate C2); Over the fresh spread of E. Coli, 100 ul of liquid culture of B. Subtiis not producing LALF or doesn’t have antibiotic resistance as a single drop.

Plate C3); At this contrivance we added B. Subtilis that doesn’t produce LALF but has antibiotic resistance over the fresh spread of E. Coli with RFP likewise. We aimed to see the affect of antibiotic resistance over E. Coli with RFP ,by using B. subtilis with and without antibiotic resistance.

Plate D1); We added supernatant of B. Subtilis with LALF as a single drop over fresh spread of E. Coli with RFP. Because at the beginning of the LALF part of B. Subtilis there was a throwing out signal and we expected death mostly here.

Plate D2); At this plate we applied 100 ul of supernatant of B: subtilis not producing LALF protein and not having antibiotic resistance over the fresh spread of E. Coli as a single drop.

Plate D3); At this plate we applied 100 ul of supernatant of B: subtilis not producing LALF protein and but that has antibiotic resistance over the fresh spread of E. Coli as a single drop.

Plates B2 and B1 (Respectively)

Plates C1 and C2 (Respectively)

Plates D2 and D1

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1: This part includes a gram positive promoter, an RBS sequence and SacB signal peptide sequence to synthesize the protein outside of the cell. This image shows that indicated part is considered as confirmed.

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2:This part includes a gram positive promoter, an RBS sequence and LipA signal peptide sequence to synthesize the protein outside of the cell. This image shows that indicated part is considered as confirmed.

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3: This part includes a constitutive promoter, an RBS sequence and Tat signal peptide sequence to synthesize the protein outside of the cell. This image shows that indicated part is considered as confirmed.

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4:This part includes a IPTG inducible promoter, an RBS sequence and Tat signal peptide sequence to synthesize the protein outside of the cell. This image shows that indicated part is considered as confirmed.

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5:Our “LALF (limulus anti-lipopolysaccharide factor)” protein allows stopping any kind of gram negative bacteria growth by binding their cell wall material, LPS. This image shows that indicated part is considered as confirmed.

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6:Our “reflectin” protein has the ability to reflect the light by changing its wavelength, thus its color. In our project, we planned to use this protein as an indicator on E.coli whether our LALF protein works properly or not. This image shows that indicated part is considered as confirmed.

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8:This part is a well-studied gram positive promoter. We planned to use this gene in the case of our signal peptide sequences do not work. When the protein, which is attached to this gene, is produced, we planned to blow up the bacteria; therefore synthesized protein would be in supernatant. This image shows that indicated part is considered as confirmed.

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500: To perform cloning perfectly, we used pSB1C3 in E.coli. This vector is also the main vector for our parts that works only in E.coli. All of our parts are also inserted into this vector; because it is declared that all parts must be sent to Registry in pSB1C3. This image shows that this part is confirmed.

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501: To perform ligation procedure appropriately, we designed an alternative part for all of our material parts. We inserted our promoters and proteins into pSB1C3 in order to have the alternative part that possesses different antibiotic resistance, comparing to original vectors of the parts. Moreover, it is wanted that all parts must be sent to Registry in pSB1C3. This part includes a gram positive promoter, RBS sequence and SacB signal peptide sequence. In the image, it can be seen that this part is confirmed.

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502: To perform ligation procedure appropriately, we designed an alternative part for all of our material parts. We inserted our promoters and proteins into pSB1C3 in order to have the alternative part that possesses different antibiotic resistance, comparing to original vectors of the parts. Moreover, it is wanted that all parts must be sent to Registry in pSB1C3. This part includes a gram positive promoter, RBS sequence and LipA signal peptide sequence. In the image, it can be seen that this part is confirmed.

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505:To perform ligation procedure appropriately, we designed an alternative part for all of our material parts. We inserted our promoters and proteins into pSB1C3 in order to have the alternative part that possesses different antibiotic resistance, comparing to original vectors of the parts. Moreover, it is wanted that all parts must be sent to Registry in pSB1C3. This part includes the sequence of our LALF protein. In the image, it can be seen that this part is confirmed.

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506:To perform ligation procedure appropriately, we designed an alternative part for all of our material parts. We inserted our promoters and proteins into pSB1C3 in order to have the alternative part that possesses different antibiotic resistance, comparing to original vectors of the parts. Moreover, it is wanted that all parts must be sent to Registry in pSB1C3. This part includes the sequence of our reflectin protein. In the image, it can be seen that this part is confirmed.

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800: In our project, we planned to use gram positive bacteria to synthesize our LALF protein in order to stop gram negative growth. On the other hand, to identify effectively whether our protein works or not, we decided to use reflectinprotein and to produce it from both of our bacteria; B.subtilis and E.coli. Thus, we determined to use a shuttle vector that works both bacteria kinds. This vector has ampicillin resistance for E.coli and chloramphenicol resistance for B.subtilis. In the image, it can be seen that this part is confirmed.

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900:In our project, we planned to use gram positive bacteria to synthesize our LALF protein in order to stop gram negative growth. On the other hand, to identify effectively whether our protein works or not, we decided to use reflectin protein and to produce it from both of our bacteria; B.subtilis and E.coli. Thus, we determined to use a shuttle vector that works both bacteria kinds. This vector has chloramphenicol resistance for both of bacteria kinds. This part also includes an RFP sequence. In the image, it can be seen that this part is confirmed.

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545: This part is designed; because it is wanted that all parts must be sent to headquarters in pSB1C3 vector. It possesses a promoter that works in gram negative bacteria, Tat signal sequence in order to synthesize the protein outside and LALF protein gene. In this image, indicated part is considered as confirmed.

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815: In order to stop gram negative bacteria growth, our LALF protein must be synthesized by another type of bacteria, gram positive. Thus, we designed a special gene that works in only gram positive bacteria by inserting a promoter specialized for gram positive. Also, SacB signal sequence that allows us synthesizing the protein outside and LALF protein gene is included.All components are inserted into 800 that isdesigned for gram positive. In this image, indicated part is considered as confirmed

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915: In order to stop gram negative bacteria growth, our LALF protein must be synthesized by another type of bacteria, gram positive. Thus, we designed a special gene that works in only gram posit