Team:Fatih Turkey/Experiments

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 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 900 that is designed for gram positive. In this image, indicated part is considered as confirmed.

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925: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, LipA signal sequence that allows us synthesizing the protein outside and LALF protein gene is included.All components are inserted into 900 that is designed for gram positive. In this image, indicated part is considered as confirmed.

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516: This part is designed; because all parts must be sent to headquarters in pSB1C3 vector. In this image, indicated part is considered as confirmed.

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526: This part is designed; because all parts must be sent to headquarters in pSB1C3 vector.In this image, indicated part is considered as confirmed.

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536:In our experiments, we aimed to indicate whether our LALF protein works or not, we used an alternative indicator protein, reflectin. This part is designed to work in gram negative E.coli. A constitutive promoter, a Tat signal sequence that allows synthesizing the protein outside and reflectin protein gene is included in pSB1C3. In this image, indicated part is considered as confirmed.

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546:In our experiments, we aimed to indicate whether our LALF protein works or not, we used an alternative indicator protein, reflectin. This part is designed to work in gram negative E.coli. An IPTG inducible promoter, a Tat signal sequence that allows synthesizing the protein outside and reflectin protein gene is included in pSB1C3. In this image, indicated part is considered as confirmed.

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596:In our experiments, we aimed to indicate whether our LALF protein works or not, we used an alternative indicator protein, reflectin. We designed an alternative part in the case that our signal sequences do not work. This part includes a promoter that works in only gram negative and reflectin protein gene. All components are inserted into pSB1C3. In this image, indicated part is considered as confirmed.

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Experiment of liquid culture

   We wanted to see the effects of supernatant of B.subtilis which produces LALF in order to stop e.coli with RFP growth in liquid culture.
   We put 8 ml LB broth in falcons.
   We planned 3 control groups.These are:

•       Control group 1:Includes only 5ul e.coli which waited for 12 hours in 37⁰C. These can synthesize RFP.
•       Control group 2:Includes only 5 ul b.subtilis which produces LALF. They waited for 12 hours in 37⁰C.
•       Control group 3:Includes only 5ul b.subtilis which do not produce LALF. They waited for 12 hours in 37⁰C.

   First of all, we put 5ul e.coli which has RFP into 8 falcons as experimental groups. We waited experimental falcons for 12 hours in 37⁰C . Then, we added 1ul,10ul,100ul and 1000ul liquid culture with LALF and liquid culture without LALF. After this, we incubated them in 37⁰C and measured their OD value 2-4-8-12 hours later in 405 nm. Their results are as below: