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 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.

Plate A1 and A2

We ensured the formation of Bacillus Subtilis biofilm with LALF(plateA1) and without LALF(plateA2).Then we put onto E.coli with RFP this biofilms.Obviously we observed E.coli with RFP can’t grow on LALF biofilm.

Plates B2 and B1 (Respectively)

Plates C1 and C2 (Respectively)

Plates D2 and D1

DISC EXPERIMENT 2

We have prevented E.coli growth in our experiment. We took antibiotic disks which are used in hospitals.

We put antibiotic disks onto spreads of E.coli which produces RFP protein and has the chloramphenicol resistance. Then, we added different amounts of supernatant.

We used supernatant with LALF and without LALF of B.subtilis. We centrifuged LB broth culture which was incubated for 12 hours at 37°C at 7000 rpm for 5 minutes. It is filtered with 0,2 filter, afterwards.

E.coli with RFP was spread on both of two plates. 40ul, 80ul, 120ul and 160ul supernatant of B.subtilis which do not include LALF protein was added into left plate. Then 40ul, 80ul, 120ul and 160 supernatant of B.subtilis which include LALF protein was added into right plate .We got results which support other experiments.

 

Supernatants with and without LALF protein was put onto 50 ul spread of E.coli with RFP in declared amount at the picture .Again we got results which is overlapped other our experiments.

The reason of making this experiment with antibiotic and without antibiotic plate was if antibiotic effects LALF or not?

Sterilization period of Supernatant

We have deduced that supernatant of B.subtilis that produces LALF sterilize the surfaces that LALF had applied on. Thus, we wanted to test how long this sterilization exists. To do this, supernatants with and without LALF waited in +4 ⁰C for 24 and 36 hours. Then, we tested their ability to sterilize.

Equal amount of E.coli with RFP sequence were spread into the fresh plates Then, 100 ul supernatant with and without was added on plates. Note that no colony grew on where supernatant that contents LALF was added after both 24 and 36 hours. (the plates on the right)

To understand better how much effective our protein is, we measured the diameter of the no growth zone. In 24 hour waited plate, diameter was 2,5 cm; on the other hand, it was measured 1,7 cm in 36 hour waited plate.

 

THE SUICIDE EXPERIMENT OF E. COLI

Our K541545 part contains IPTG promoter and the gene part of LALF protein on a backbone which has resistance to chloramphenicol. We transported this part to E. coli for testing whether E. coli would kill itself or not.

Experiments of plates with and without IPTG

Plate A1: We streaked E coli culture which has K541545 on this plate and the plate includes IPTG and Chloramphenicol.

Plate A2: We streaked E coli culture which has K541545 on this plate and the plate includes just Chloramphenicol.

Plate B1: This plate does not have IPTG and we streaked E.coli culture which does not have IPTG promoter.

PlateB2: This plate has IPTG and we streaked E coli culture which has IPTG promoter.

We prepared the B1 and B2 plates for testing toxic effect of IPTG. Because we wanted to be sure that the only factor affecting growth of E coli is LALF protein.

 

THE EFFECT OF FENTON REAGENT SOLUTION ON THE BIOFILM

Fenton Reagent solution is a solution that can kill the B. Subtilis spors and bacteria. It kills the spors by oxidizing with its Cu ions.

We prepared this solution in two ways one with the hydrogen peroxide (OFR) according to the literature and the other with distilized water (DFR). In our experiments we learned which of these two solutions is more effective on E. Coli, B. Subtilis or the the spores of B. Subtilis and degree of its effectiveness.

The aim of this experiment is seeing the effects of the fenton reagen solution on the B. Subtilis and E. Coli on the biofilms.

We prepared Corn Strach Lb Agar for a more qualified biofilm. In order to understand the death in the plates we added a small amount to the liquid culture.

Plate 1: On this plate OFR is added on the B. Subtilis biofilm

Plate 2: We formed another B. Subtilis biofilm and added same amount of DFR on it.

Plate 3: On this contrivance one drop of E. Coli with RFP on the B. Subtilis biofilm and added same amount of DFR on it

Plate 4: On this contrivance one drop of E. Coli with RFP on the B. Subtilis biofilm and added same amount of OFR on it

Plate 5: this plate prepared as a control group of 3^rd and 4^th plates. On this contrivance one drop of E. Coli with RFP on the B. Subtilis biofilm but any kind of fenton reagent solutions didn’t add on it.

RESULT: After adding the FR solutions on all of plates, we measured their OD values in the end of this experiment we measured again and saw that the 1^st, 2^nd, 3^rd, 4^th plates OD values didn’t change. But the the OD value of 5^th plate increased. 5^th plate is the control group.

 

 

Figur2: plate 3 and plate 4

The Difference between Colonies Containing and Not Containing Gene Parts of Reflectin Protein

We transfered the plasmid which includes the gene part of reflectin protein (j04450 shuttle, j04500 backbone, and 1006) to E. Coli and we saw that there is a difference between reflectin including colonies and the colonies which does not include reflectin protein.

We saw that the plate which does not include reflectin protein is normally transparent. But the other plate which includes reflectin protein seemed white. And this is showing us that our plasmid is working and the protein is produced by E. coli.
Figure-1: the difference of colonies is showing clearly, the colonies at the right side are seemed white and the colonies at the left side are seemed transparent.

Additionally, the colonies are looked into by microscope. The colony which is including reflectin has some transparent points. But the control group hasn’t these points. We are thinking that because of reflectin protein there are the points in these colonies.

 

Figure-4: Image of the colonies which are include reflectin protein by microscope it’s clearly showing that the transparent points in the colonies

 

Figure-6: Image of the control group which is not including the 1006 gene part and reflectin protein and it can clearly showing the difference from the colonies which include reflectin protein. There aren’t the transparent points here.

Consequently, it’s confirmed that the 1006 gene part is producing the reflectin protein

 

We wanted to investigate to effects of supernatant includes LALF protein to E.coli in this experiment.

Therefore we put 8 ul LB broth into falcons.then we added them E.coli which has rfp and incubated for 12 hours in 37⁰C .We put pure E.coli into only one falcon for control.We added 1ul,10ul and 100ul supernatant with LALF and without LALF.

We diluated them 7 times 2-4-8-12hours later then we spreaded 10ul to plates which has 1/0,5 l Cloramphenicol.We did santrifuge liquid culture which incubated for 12 hours at 37⁰C at 7000 rpm for 5 minutes because of preparing supernant with LALF and without LALF.Then we passed them from the 0,2 filter.

Plates were spreaded after 2 hours later:

1a)We added 1ul supernatant includes LALF into left plate and 1ul supernatant without LALF into right plate

1b)We added 10 ul supernatant includes LALF into left plate and 10 ul supernatant without LALF into right plate

1c)We added 100ul supernatant includes LALF into left plate and 100ul supernatant without LALF into right plate

Plates were spreaded after 4 hours later;

2a)We added 1ul supernatant includes LALF into left plate and 1ul supernatant without LALF into right plate.

2b)We added 100ul supernatant includes LALF into left plate and 100 ul supernatant without LALF into right plate.

Plates were spreaded after 8 hours later;

3a)We added 1ul supernatant includes LALF into left plate and 1ul suoernatant without LALF into right plate.

3b)We added 10 ul supernatant includes LALF into left plate and 10 ul supernatant without LALF into right plate.

3c) We added 100ul supernatant includes LALF into left plate and 100 ul supernatant without LALF into right plate.

Plates were spreaded after 12 hours later;

4a) We added 1ul supernatant includes LALF into left plate and 1ul suoernatant without LALF into right plate.

4b)We added 10 ul supernatant includes LALF into left plate and 10 ul supernatant without LALF into right plate.

4c)We added 100ul supernatant includes LALF into left plate and 100 ul supernatant without LALF into right plate.

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:

Graph1 : Absorbance(OD405) value difference through time of AntiLPS-LPS binding with 1 ul LALF

Graph2 : Absorbance(OD405) value difference through time of AntiLPS-LPS binding with 10 ul LALF

Graph3 : Absorbance(OD405) value difference through time of AntiLPS-LPS binding with 100 ul LALF

Graph4 : Absorbance(OD405) value difference through time of AntiLPS-LPS binding with 1000 ul LALF

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CONFIRMATION

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.

CHARACTERIZATION OF LALF PROTEIN

 

TARGET: Showing that the LALF protein is produced.

 

1)

 

 

 

Transformation is done and green colonies are selected then we started to do our second procedure.

 

 

2)

 

 

 

TARGET: The characterization of part 545 which is done suicide experiment of E.coli

1) The sequence of part 545

Procedures of liquid culture which is planted by cloning team:

-PCR

-Existence of expected plasmid is confirmed by electrophoresis.

-Sequence

2) Part 545 c DNA sequence

Procedures of liquid culture which is planted by cloning team:

Plasmid isolation

BL21 transformation

Liquid culture

RNA isolation

C DNA

PCR

Producing of expected part’s mRNA is confirmed by electrophoresis.

Sequence

 

RESULT: LALF find in the DNA sequence and mRNA sequence. We produced LALF protein by E. coli.