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Team:IvyTech-South Bend/Notebook
From 2010.igem.org
Contents |
Notebook
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8/24/10
Today we will be pouring new plates for streaking new transformed cells.
8/27/10
We will be running a gel to determine if our DNA sample was successfully electroplated.
8/27/10
Today we will be electroplating part KBBa_3131010
8/31/10
Results of transformation - the plates had growth but (no) distinct colony pattern.
8/31/10
After agar is dissolved, and allowed to cool, I will add Amp in different doses to specific plates. JH
9/1/10
Today the team will be making up the same plates as I made yesterday. Professor T. wants new Amp stock made to document how much is used into the stock.
9/2/10
Results from streaking –
9/2/10
Protocol For Making LB-Broth/Agar
9/3/10
Today I’m finishing making the LB/Agar from yesterday.
9/9/10
Due to conflict we will be changing from E Coli to yeast.
9/10/10
Lux Casette Right Promoter BBa_I1051
9/14/10
Today we found growth from our Electroporated E – Coli parts
9/15/10
- Today we will be extracting DNA from out electroporated cells/T9002
9/16/10
Today we will wash our electrocomp grown t/pos bacteria with 1.2 mL Glycerol then electroporate Following protocol from page 15 First prof. T sterilized the glycerol passing it through the .2 micron filter into 50 mL sterile centrifuge tube 1 – then we added 1 mL 10 percent Glycerol to suspended cells 2 – then centrifuged at 1000 rpm for 10 min - then removed supernate with BR 1000 micropipettor - then add 1 mL 10 percent Glycerol then repeat steps 1 & 2 “4” times then remove sup. and freeze cells at -80 degrees C freezer.
- Today we also set up a new IGem work are in back lab look at pic
9/17/10
Today we will be doing a DNA Extraction from/of part T9002 for electroporation this will determine if we have a stable host for our beast this will also Work!
Following protocols from pgs 15 &19 -instead of Electroporating our part we will use PG10 to determine if these lines are good hosts
First we will be pouring a full pack of LB/Agar/Amp plates using the LB/Agar I made on 9/2/10 then adding amp made by DG on 9/1/10 we will also add 5 mL Arabonse to the Agar before pouring plates have been poured now letting stand for an hour to harden.
-Now I will be making 1L of LB/Broth for Culturing. Lot 9082989 ex. 2014-02-28 BD Difco LB/Broth – Lennox 20 g per 1L so I will put 10 g into 2 elenmyier flask/capped one weighed 10.0023 g and the other weighed 10.0011 g then I added 500 mL into each flask then shook to mix then dissolved in microwaved for 8 oz setting once then placed into Autoclave for 15 min @ 121 degrees C until done.
Now we are going electroporate our grown t/pos Electro Comp Cells from yesterday with PG10 using protocol from pg 15 changing # 5 to 5 mL of PG10 We will be using a different protocol for electroporation of grown t/pos bacteria from Bio-Rad’s website prof. T will print and I’ll place here.
The mycobacterium will be done by electroporation protocol on pg 15.
1) First I placed a Cuvette into Ice bath and thawed electro comp cells 2) I set up 5 1.5 mL Sterile Centrifuge tubes with 900 mL SOB media 3) we will electroporate Irene’s electrocomp cells w/PG10 and mine will contain the parts after electroporation.
Code AT – A.tumefacions SL – S.lactis BM – B.magetanium MP – Mycobacteria BT – B.thurigensis
split cells using 40 mL Hy part my cells then electroporate
I electroporated all except mycobacteria now letting cells sit for at least Just added 1 mL to electroporated Cultures they had sat for 20 min before adding.
- Now I will electroporate mycobacteria using E – Coli protocol from pg 15 using 40 mL cells 4 mL pg10 Electroporation hit twice Read out 1.8 kv @ 5.2 ms then 1.79 kv @ 5.1 ms. then placed into 900 mL SOB media then incubate @ 30 degrees C for 3 hrs.
Results From this we found that agrobacteria will be the best host for our beast/IGem we will do further testing towards our ultimate goal.
9/21/10
Today we will electroporate Agrobacterium with part T9002 using protocol from pg 22 – 23
1) I will put frozen electro comp cells from -80 degrees C freezer 2) We will do two “2” electroporations with (AT) one with 5 mL T9002 Gene, and one with 20 mL 3) Icing Cuvette (.1) and now prepared to electroporate 4) Reading for 20 mL was 2.2 kv @ 0.70 ms 5) then removed with 1 mL SOB media 6) Placed into 1.5 mL Sterile Centrifuge tube at room temp for 3 hrs prof T. will streak.
9/22/10
Today we have growth from electroporation done yesterday we are going to test by hitting with (AHL) the 20 mL grew not 5 mL sample 1) Scrap bacteria from transformed plate 2) Set up (3) Cuvettes 1 with 200 mL of bacteria + 800 mL LB – Lennox broth 2 with 200 mL of bacteria + 800 mL of A.tumafaciens superant 3 with 200 mL of bacteria + 800 mL of E – Coli Superant
4 with 200 mL LB broth + 800 mL of LB
5 with 200 mL LB broth + 800 mL of A tumafaciens
6 with 200 mL LB broth + 800 mL of E – Coli Superant change 500 mL cells either Agro-T9002 for sample A,B,C or Agro for D,E,F and 500 mL Sup + 200 mL SOB
9/22/10
The only one that should glow should be (C)/(3) E – Coli w/transformed part in LB For Supernate of A.Tumafaciens we will use suspended T9002 in agrobacteria prof T. set up yesterday as our 200 mL spl. - Today I will be electroporating part #’s I732094 (Lacz) – GFP, and part # F2620 (LuxR) into E – Coli Electrocomp cells. DG pulled the DNA parts from the Bio Brick and placed into 50 mL Electro comp E – Coli cells and 5 ml of DNA spls. now we will electroporate using protocol from pg 15 We recovered cells shooting 900 mL into Cuvette then drawing it off with electroplated cells. Placed at room temp to grow for 1 hr then place and suspend in LB/Amp (10 mL) - I took our streaked to try to get an Isolated Colony Streaking and Heating between each pass. then placed into incubator. - Results after letting incubate for 2 days we have found that the incubator was set to too low of a temp we raised the temp to 37 degrees C from 26 degrees C we will see where we are at on Tuesday.
9/23/10
Today I have been setting up for the trial of our part T9002 inside agrobacteria to see if in the presence of AHL it floureces.
9/24/10
Today I’m testing absorbance of our trials @ 395nm
9/28/10
Today we will be picking 2 colonies from the Lacz, LuxR electroporated plates that were streaked on 9/22/10
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