Team:Fatih Turkey/Notebook

From 2011.igem.org

deneme baslik

Week:

14.07.11

PROBLEMS AND SOLUTIONS

# 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

SITUATION
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

16.07.11



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.
Fourplateswerealsopreparedalternativelybystreaking.
6.       500 ul FR withdistilledwaterwas put in 1,5 ml Eppendorf; exposedtoheatshock at 50 C hopingthat it wouldreflectblue-greencolorwhichindicatesthesoluitonlosesitskillingactivity. Itdid not happen.
http://igem.org/wiki/images/d/da/16%2C07%2C2011.pdf

20.07.11



http://igem.org/wiki/images/7/7f/20_07_11.png
http://2011.igem.org/wiki/images/4/41/Dcd.png
http://2011.igem.org/wiki/images/6/64/Vs.png
http://2011.igem.org/wiki/images/b/b7/Fg.png

DISCUSSION

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.