Team:Fatih Turkey/Notebook


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deneme baslik

JULY 2011:




# Prepare to competent materials;
*We prepared the competent materials for tomorrow
(tomorrow we will prepare the competents of b. subtilis and it will be 5th time:))
*we prepared our b subtilis plates for using tomorrow
*we have two plates for competent, one of them is wild type and other stain is ATCC6633
*we named them not to confuse, the ATCC663 strain's new name is Ahmet, wild type bacteria's new name is Mahmut :)
those names are turkish male names

# Transformation;
*we couldn't get the expected results at transformation of b. subtilis in this week today we will try to make the most succesful transformation ever :)
The Problems and The Solutions:
-We decided to some changeings at our procedure of competent
*incubation time will be a hour, firstly we will add the DNA to b. subtilis pellet without medium B then we will shake the mix for 30 min. and we  will add medium B on this mix than we will shake new mix again for 30 min.
*amount of DNA will be increased

Transformation; we prepared 4 plates for transformation
1. plate : b. subtilis pellet + 6 microliter plasmid ---> incubation/shaker30 min.--> adding 100 microliter medium B --->incubation/shaker30min. --->spreading 5ug/ml Chm plate
2. plate: b subtilis pellet + 10 microliter plasmid ---> incubation/shaker30 min.--> adding 100 microliter medium B --->incubation/shaker30min.----> spreading 5ug/ml  Chm plate
3. plate: b subtilis pellet + adding 100 microliter medium B ---> incubation/sahker 30 min.--->adding 10 microliter plasmid ---> incubation/shaker30min.----> spreading 5ug/ml Chm plate
4.plate: b subtilis pellet + adding 100 microliter medium B ---> incubation/sahker 30 min.--->adding 6 microliter plasmid ---> incubation/shaker30min.----> spreading 5ug/ml Chm plate


1.       Plasmidisolationwasdone. (K090402/01)
2.       Competentcellpreparationwasaccomplished. (Mahmut)
a.       Three Eppendorfswereexposedtotransformationprocess.
b.      NineteenEppendorfswerestocked in -80 C byglycerolstockprocedure.
3.       Onemediabottle LB (200 ml) wasprepared.
4.       Fourteenplateswithoutantibioticswerestocked.
5.       Fentonreagentexperimentwastested.
a.       Twofalcons(50 ml) thatincludedoneE.colicolonyperfalconwereprepared. Exposedtoincubation at 37 C.OD650 of falconsweremeasured as 0,37 and 0,08. Theonethat has bigger OD650 wasseperated as 1 ml culturetofourEppendorfs.
b.      Oneepp. wasindicated as control. OnewasincludedFentonreagent (FR) dissolvedoxygen, onewasincluded FR withdistilledwaterandlastonewasincluded x10 FR withdistilledwater.
c.       All of theEppendorfswereexposedto FR solutionsfor 30 minutes.
d.      FourplateswerepreaparedbyspreadingperEppendorfsthat had beenprepared in step 5b.
6.       500 ul FR withdistilledwaterwas put in 1,5 ml Eppendorf; exposedtoheatshock at 50 C hopingthat it wouldreflectblue-greencolorwhichindicatesthesoluitonlosesitskillingactivity. Itdid not happen.



As a conclusion we observed Distilled water including Fenton Reagent (DFR) is more effective than Oxygenated water including Fenton Reagent (OFR) in E.coli. When 25 uL OFR and DFR have been added in E.coli cultures,  82% of DFR treated culture is dead and 47% of OFR treated culture ‘s dead. We didn’t see any colony which included 50 uL and more DFR.  However, after 75 uL and more addition of OFR, any colony formation has not been observed.
On the other hand we observed OFR is more effective than DFR in Bacillus subtilis.When 10 uL OFR and DFR have been added in Bacillus subtilis cultures,38% of DFR treated culture is dead and 98.3% of OFR treated culture’s dead. Not only the 25 uL OFR treated but also 25 uL DFR treated culture is dead.
If we want to compare E.coli and B. subtilis, we can say that Fenton Reagent application is more fatal for Bacillus subtilis. Both 25 uL OFR and DFR treated Bacillus subtilis culture is dead but 25 uL OFR and DFR treated E.coli culture is still alive.

  5 mL lb which include E.coli has been prepared in the morning then when the culture grown up it has been stroke in lb-agar which contain corn starch and no antibiotics.
The wiki arrangement has been kept on.
The fridge has been tidied up.
400 mL lb-agar with ampisilin has been prepared.
E.coli which have GFP, RFP and CFP has been stocked in glycerol. At the same time this cultures have been spreaded.
Sporocide experiment has been done.





29. 07.11



-K(J04450)+Shuttle(K090403) 1/2/3


DIGASTION,LIGASTION (with pst and ecoR1)


we prepared those plasmids with amphiciline.

-1001+1005+K (only plasmid isolation)
we prepared those plasmids with chloramphenicol





1001+K  C( C is the name in glycerol stock)
1002+K  C
1003+K  A
1004+K  A
1005+K  A
1001+1005/sht  BG
K143053+1006/sht  BG
                >>>>>>All of them have been isolated and digested with EcoR1, Pst1 then  made electrophoresis for confirmation.
We have growned another some colonies from 1002+1006/sht and 1001+1006/sht (which grown in two antibiotics before) overnight (yesterday) and today they have been stroke which contain two antibiotics  in the morning and their falcons have been kept in the fridge .
     >>>which picked colonies  x ,y ,z ,t   from A plate
>>> which picked colonies   Ax , Bx, Cx , Dx , Ex  from B plate
    >>>which picked colonies  Ax , Bx , Cx , Dx  from A plate
>>> which picked colonies  x,y,z,t,s  from B plate

  In the evening we observed  1001+1006/sht     By,  ADx , Bs streaks didn’t grow so we put in the new liquid culture from their morning’s liquid culture for glycerol stock. Also we prepared second  new liquid culture By for it’s plasmid isolation and confirmation.
1002+1005/sht  C have been taken from glycerol stock and put in liquid culture for plasmid isolation. (it’s necessary for bacillus transformation to our DISC test)



Liquid Culture: A,B,C

  Yeni Competent için daha önceden kendi yaptığığmız competent E.coli lerden sıvı kültüre koyduk




J04500+1006+K      K,L,M,N,P
1001+1005+K          K,L,M,N,P
1002+1006+K          K,L,M,N,P 
    >>> >>>>>>all of them have been isolated which put in liquid culture from yesterday. But plasmid isolation kit’s coloumn finished.So we continued with gel extraction kit’s coloumn and it’s amount which in gel extraction protocol.Then we used electrophoresis.However we didn’t observed any clear and right band in electrophoresis.This may be because of our using different coloumn and protocol.So we didn’t make gel extraction and transformation.
We put them again liquid culture with Chl which choose different  colony called X, Y, Z, T, V .And also we put   1004+1005+K  B ,     1002+1005+K  A ,   1002+1005+K B ,    1004+1006+K  C,   1001+1006+K  A  in liquid culture with Chl which confirmed in electrophoresis and took with  gel extraction.



Liquid Culture:
1001+1005+K MG
1001+1005+K SNG
J04500+1006+K FT
J04500+1006+K AT
1001+1005+K İG

1004+1005+K B
1001+1006+K A
1004+1006+K C
1002+1005+K B
1002+1005+K A
1001+1005+K MG
1001+1005+K SNG
J04500+1006+K FT
J04500+1006+K AT
1001+1005+K İG


1.       Ten colonies that hadparts with promoter, RBS, signal sequence and our special genes were incubated in the ampicilin and chloramphenicol rich medium. The plates that had no colonies incubated were ours which we had expected.  These were 1001+1005+K/TNG, 1001+1005+K/HNG, J04500+1006+K/BT, J04500+1006+K/ST and J04500+1006+K/FG.
2.       Ten colonies were incubated in the ampicilin and chloramphenicol rich LB and five additional colonies were incubated in the only chloramphenicol rich medium in order to eliminate by observing if any colony would be seen or not. J04500+1006+K ST, 1001+1005+K TNG, 1001+1005+K HNG, J04500+1006+K FG, J04500+1006+K BT were not seen in the falcons. (These are confirmed.) However; 1001+1005+K MG, 1001+1005+K SNG, J04500+1006+K FT, J04500+1006+K AT, 1001+1005+K İG were not confirmed.
3.       Plasmid isolation was done for 1004+1005+K B, 1001+1006+K A, 1004+1006+K C, 1002+1005+K B and 1002+1005+K A.
4.       Glycerol stock was performed for 1004+1005+K B, 1001+1006+K A, 1004+1006+K C, 1002+1005+K B.
5.       1002+1005+K/1, 1002+1005+K/2, 1002+1005+K/3, 1002+1005+K/4 and 1002+1005+K/5 were put into LB medium for incubation and spread on the plates.
6.       Digestion-ligation was performed for shuttles. (except for 1002+1005+K/A)
7.       Electrophoresis was done for confirmation of J04500+1006+K BT, J04500+1006+K FT, J04500+1006+K ST, 1001+1005+K HNG, 1001+1005+K TNG, 1003+1006+K, k143053+1006+K and its shuttle. For all genes, gels were cut and stocked in +4 C.







We tried to different amount for  addition up, down, destination  part plasmid  in ligation protocol. We did that for increase possibility of exist our part in backbone.
1002 1005 can lig

1002 up>>3 uL
1005 down>>3 uL
can>>1 uL
dH2O>>10 uL

1002 1005 sht lig

K+sht>>1 uL
1002 1005 K1Y>>5uL
dH2O>>11 uL
1002 1005 K1X dig
10 uL plasmid            >>>>>It’s liquid culture can’t pass double antibiotic test but we did it’s double EcoR1,Pst1                             digestion  for see what can be plasmid in this culture 32.5 uL dH2O
Digestion which show below, has been made for confirmation.
K+sht dig
4.2 uL plasmid
38.3 uL dH2O

1002 1005 K dig.
7.5 uL plasmid
35 uL dH2O

Can dig.
4.7 uL plasmid
37.8 uL dH2O



These are passed from couple antibiotic experiment which made yesterday:
*K143053+1006+K A1
*K143053+1006+K A2
*K143053+1006+K  A3
*K143053+1006+K  A4
*1001+1005+K  X
*1001+1005+K  Y
*1001+1006+K  B1
*1001+1006+K   B2
*J04500+1006+K   B3
*J04500+1006+K   B4
*1003+1006+K    A1
*1003+1006+K A4
*1004+1006+K   B1
*1004+1005+K  B2
*1002+1006+K   B4
We made plazmid isolation , digestion(ecori1_ pst)and electrophoresis ..




Transformasyondan sonra sıvı kültüre konulan K541_596 (222)in
plasmid isolation u yapıldı jel de yürütüldü fakat iyi görüntü alınamadı.
Nedeni 12ml lik jel hazırlanmasından ya da içinde çok çeşitli plasmid olmasından dolayı olabilir.
K541_925A nın plasmid isolation u yapıldı. Jelde yürütüldü.









1.       For each815, 815 gex, 825, 825 gex, 826, 926, 915, 915 gex, 925, 925 gex two colonies had been chosen and all were found wrong; thus this procedure is replied.
2.       The parts above are isolated from their LBs.
3.       Same parts are then digested with Xba1 and Pst1.
4.       Same parts are loaded into gel.
a.       For 915 and 915 gex, destinationplasmids were foundwrong; but genes were correct.
b.      We were not so sure for destination plasmid of 826; but its gene were also correct.



1.       815 ep, 815 xp, 915 ep, 915 xp, 915 gexxp, 515 xparedigested-ligated. (Note that transformation is not performed because of absence of prepared compotent cell.)
2.       545, 504-GFP and 915 aretransformated.
3.       Each for 596, 596-b, 546, 536; two colonies are chosen. LBs for these parts are prepared. (8 LBs)
4.       For 815, 815 gex, 915 and 915 gex; four additional LBs are prepared in order to gain more copies of them.
5.       An antibiotic free LB is prepared to make compotent cell next day. One compotent cell in the stock is transferred into it.


1.    We made  plasmid  isolation to 536H,546H,596B_G,596B_H,596H , j04500 but unfortunatelly  we fixed 596 G and 546 G in experiment  time .With xba1 and pst1:
536H   >>>>                           13.1                                              29.4
546H >>>>>                            7.4                                               35.1
596B_G >>>>                             15.1                                           27.4
596B_H >>>>                       12.5                                                30
596G+546>>>>                     11.6                                                  30.9
596 H >>>>                             16.1                                                26.4
With ecor1 and spe1:
J04500_2(double dig.for up) >>>>         6.6                                                  35.9
With e.cor1:
J04500_2(single)>>>>     6.6                                                                       36.9
J04500_2(undigest)>>>>   6.6                                                               37.9(WE FORGOT  THİS!!!    So we didnt  made electrophoresis j04500 _2 single and undigest and didnt put all of  j04500 _2(double dig.for up  )  to gel.
2.then made them electrophoresis.
3. 815 ,815 gex,545,916,j04450,can are  put liquid culture from glyserol stock. We choose  2 colonies from 915 gx xp   and also 2 colonies  from 504 gfp then put all of them 10 ml liquid culture.
4.Top 10,DH 5alfa ,PET 28 a in BL21,BL 21 are put 10 ml liquid culture.


1.       For each of 915 gexxpand 504-gfp, two colonies were incubated in 10 ml LB.
2.       916, 900, 915 gexxp, 504-gfp, 545, 500, 815, 815 gex are isolated.
3.       The parts above are digested with Xba1 and Pst1 except 545, 500.The separts are remainedun digested; because both of them had been confirmed.
4.       Digested parts are loaded into gel.
a.       All parts except 900 are found wrong.


1.       Toligate 816, 825, 826, 916, 925, 926, 536 and 546; upstream, downstream and destination plasmids are digested with  appropiate enzymes.
2.       Digested solutions are loaded into gel and all of them are confirmed.
3.       For each of 815 and 915 parts in the stock; LB solutions are prepared.


1.       Because of absence of 800, digested solutions that prepared for the ligation of 816, 825 and 826 are notligated.
2.       536, 546, 916, 925 and 926 are ligated.
3.       916, 925 and 926 transformated twice. (We have two plates each of these parts.) 536 and 546 are transformated into only one plate.
4.       LBs that were prepared yesterday are prepared again. (Isolation could not take place today.)



Reporting 12,09,2011

1.       Transformation was done for 915 gex  21/06 , 915 gex 31/06.
2.       Isolation was done for 4x800, 536, 546. These parts are also digested with EcoR1 and Pst1.
3.       Digestion was also done for 500, 1, 2, 5, 6 and 900 in order to ligate these to gain 515, 525, 516, 526, 916, 925 , 926. Digested genes are loaded into gel.
4.       815, 816, 825, 826, 515, 525, 516, 526, 916, 925 and 926 are ligated.


Reporting 13,09,2011

1.       Two colonies had been chosen for each of 536 and 546. LBs of these parts are isolated and digested with EcoR1 and Pst1 afterwards.
2.       Digested parts are loaded into gel.
a.       536-ALASKA and 546-BELARUS are found correct. However; in the 536-ALASKA gel fragment, there are two unknown and unexpected bands which must be considered carefully.
3.       515, 525, 516, 526, 815, 825, 816, 826, 925, 916 and 926 could not be transformated because of lack of plates with chloramphenicol.
4.       LBs of 915 gex 2/9 and 915 gex 31/8 are prepared.
5.       915 gex 2/9, 915 gex 31/8 and 800-e are digested with Spe1 and Xba1 in order to achieve ligation without needing Pst1 because of the false Pst1 site in 800-e.


Reporting 14,09,2011

1.       915 gex 31/8 and 915 gex 2/9 are isolated.
2.       Same samples are digested with EcoR1 and Pst1.
3.       Digested parts and additionally 800e that had been digested with same enzymes are loaded into gel.
4.       For each of 815 gex, 915 gex and 916, two colonies are chosen in B.subtilis plates and LBs are prepared.
5.       LB-agar is prepared in order to perform transformation for unconfirmed parts.