Device

Part:BBa_K541515:Experience

Designed by: Cihan TAŞTAN   Group: iGEM11_FAGEM   (2011-07-13)

Disc Experiment

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.


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Plates B2 and B1 (Respectively)

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Plates C1 and C2 (Respectively)

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Plates D2 and D1


Experiment of Supernatant


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:


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


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


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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;


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


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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;


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


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


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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;


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


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


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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:

 a) Control group 1:Includes only 5ul e.coli which waited for 12 hours in 37⁰C. These can synthesize RFP.
 b) Control group 2:Includes only 5 ul b.subtilis which produces LALF. They waited for 12 hours in 37⁰C.
 c) 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 Grafi1.jpg

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

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

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

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CHARACTERIZATION OF LALF PROTEIN


TARGET: Showing that the LALF protein is produced.

PROCEDURE 1:



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J04500-GFP


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PSB1C3


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





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PROCEDURE 2:


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Green colonies are selected and picked up then next procedures are completed.

1) Plasmid isolation

2) Digestion

Existence of expected plasmid is confirmed by electrophoresis.

3) BL21 transformed

4) BL21 liquid culture planted

5) RNA isolation

6) c DNA

7) PCR

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

8) Sequents

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RESULT: Since mRNA of LALF protein has been produced, the promoter is producing both LALF and GFP sequences.

CHARACTERIZATION

TARGET: Characterization of part 915 which is used for death test

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


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TARGET: The characterization of 915 part has done and we got expected results


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