Team:IvyTech-South Bend/Notebook

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Revision as of 19:44, 7 October 2010

Notebook

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 (2008) plate C002 well 3 – C, 2007, Plate 1 18P, 2006 DNA – 1 18 – P PLux/C1 Hybrid promoter BBa_K091107 – well 8 – P plate #2 yeast promoter BBa_K105024 Yeast promoter BBa_J63005 well 4 – A Plate 2 Yesterday (9/9), I streaked 2 plates containing LB/Agar. I streaked the transformed electro comp cells using the turntable method. Today no growth was found. Possible reasons for error I might have not cooled glass hocking sticks long enough.

- Today –
We need 1 PLux, 2 Yeast Transcription Factor 3 LuxR 4 Yeast Promoter 5 Yeast promoter that responds to yeast trans factor, we must look at Igems registry

Parts list for Igem 1 PLux BBa_K091107 10,12,21,23,25 Plate 2 Well 8 – P 2 P Yeast BBa_J63005 10,12,21,23 Plate 2 Well 4 – A 3 LuxR BBa_T9002 10,12,21,23,25 Plate 2 Well 9 – A 4 Gal 4 BBa_K105007 10,12,23,25 Plate 3 Well 9 – I 5 Yeast Prom. BBa_K207001 10,12,23,25 Harvard

We are going to electroporate parts 1 – 3, 4 today using the same protocol from yesterday. 1) I cleaned the hood using alcohol and all pippettors boxes and tools. 2) Set up Ice bath and placed all tools in Hood 3) the first part I will be electroporating is part # BBa_K091107 Plate 2 well 8 – P 4) I added 10 mL 18ohm H2O to well 8 – P and pipetted up and down 4 times then removed 2 mL from well and placed into 100 mL comp cells, Gal 4 transcription factor BBa_K105007 Plate 3 well 9 – I 5) First electro was 1.8 kv at 5.2 ms. 1A) we electroporate part # BBa_K105007 from plate 3 1.8 kv 4.2 ms well 9 – I following the same protocol from page 15. 2) we electroporated part # BBa_T9002 from plate 2 well 9 – A we added 2 mL of 18 ohm H2O to resuspend the DNA spl. electroporate 1.8 kv 4.2 ms 3) Now that they have sat for 1 hr. we will streak 2 plates for each part so “6” in all 4) with remainder I will add half each tube to 10 mL LB – Lennox 3 with Amp made fresh and 3 w/out Amp and put on shaker. -Making Amp-

250 mL treats 250 mL 10 mL per 10 mL .845 g / 30 mL

I added .6909 g to 5 mL 18 ohm H2O the dissolved I added 10 mL to “3” 15 mL Sterile Centrifuge tubes then added 10 mL LB – Lennox to “6” tubes then added 400 mL electroporated cells to them part1, part 3, part 4

9/14/10

Today we found growth from our Electroporated E – Coli parts now we took 10 mL made by GM 4/10/10 and poured 10 mL into 3 sterile tubes then added 10 mL Amp to each tube, then placed a large Colony from each part into these tubes then placed on shaker until tomorrow. - We have discovered that yeast will take a bunch of trial and error so we will try and place our IGem into Grown t/pos Bacteria-

- We will be using-

-For now – I took my 10 mL Centrifuge tubes that had electroporated cells containing parts made Friday and placed into 500 mL 40 percent glycerol and 500 mL of each part and made “2” of each to be frozen as backups.

9/15/10

- Today we will be extracting DNA from out electroporated cells/T9002 - Wash our grown pos. bacteria with 10 percent glycerol to create Electro comp. cells - Then Electroporate T9002 into Elect. comp grown pos/t bacteria

First we will be extracting DNA from BBa_T9002 Electroporated Cells using this protocol.

9/15/10

I poured 10 mL LB – Lennox into 7 different 15 mL sterile Centrifuge tubes “2” for our electroporated parts T9002, K091107 with 10 mL Amp added 5 for our grown t/pos bacteria w/out Amp labeled then added one loop full of bacteria into each then placed on shaker overnight. Irene streaked our bacteria to set up for grown staining then placed into 37 degree incubation overnight.

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.

1) I took and set up new growing Cultures of T9002 and E – Coli electro comp cells placed 5 mL into 50 mL sterile tubes one with Agro w/T9002 and one with E – Coli to make electro comp cells. 2) then drew off “2” 1 mL inciments of T9002 Sol. and placed 4.5 mL of Amp w/T9002 3) now we are ready to perform trial taking Clean 1.5 mL Centrifuge tubes labeling 1 – 6 the first contains 200 mL Argo/T9002 & 800 mL LB/Broth second w/200 mL Argo/T9002 w/800 mL Argobacteria Supernate. Third w/200 mL Argo/T9002 w/800 mL E – Coli Supernate (AHL) we are looking for a glow from this one Fourth 200 mL LB Broth – 800 mL LB Broth Fifth 200 mL LB Broth – 800 mL Argobacteria Sixth 200 mL LB Broth – 800 mL E – Coli Supernate Then they will all be tested inside a florimeter. Dylan is running a Spec reading on Agrobacteria/and Agro/w T9002 to see if the concentrations are close to the same. then do the trial he found that absorbance was 4 times higher so Dylan diluted the sample of T9002 ¼ adding LB – Lennox to this gave a spec reading of .303 and org. was .333 a difference of about 10 percent so we believe that will be ok we will work with this . See Dylans Lab notebook for more accurate details.

for now I will be making and washing electrocomp E – Coli cells to be frozen 1) spinning off supernate from cells in 50 mL Centifuge 2) then add 1 mL 10 percent glycerol and remove place in 1.5 mL centrifuge tube I will split cells into 6 centrifuge tubes adding 1 mL 10 percent glycerol and spin inside cold centrifuge @ 1000x for 10 min. 3) then repeat a total of 4 times 4) then leave about 40 mL of glycerol into the tubes and freeze @ -80 degrees C until needed.

I took the electro comp cells I’m about to wash and ran a spec read I took 20 mL of E – Coli cells and placed into 980 mL of 10 percent glycerol then vortexed to mix then pipetted on absorbance of .15 50 20 mL into 1000w 1000/.20 = 50 X .15 = 7.5 so our OD is 7.5 mL so what I did was split the cells into 6 seperate tubes to be washed following protocol from pg20 while Dylan runs the Trial on T9002 from pg25 For results see pg 30

== 9/24/10 ==

Today I’m testing absorbance of our trials @ 395nm 1) .733 @ 395nm .678 @ 395nm 2) .855 @ 395nm .586 @ 395nm 3) .892 @ 395nm .693 @ 395nm 4) .708 @ 395nm .733 @ 395nm 5) .700 @ 395nm .700 @ 395nm 6) .693 @ 395nm .729 @ 395nm Blank) .001 @ 395nm .001 @ 395nm

1) I will blank the spec using LB/Lennox made by GM on 4/9/10 2) –Change- I blanked using the LB – Lennox Broth w/200 mL of SOB media – both made by me. 3) I took and used the same Cuvette for every spec scan washing with DI water between every trial 4) Placing about 1 mL every trial 5) Set spec to 395nm we believe that their was too much absorbance in trials 4,5, & 6 so we will do same procedure using florimider. Results say that the GFP is not being expressed in the presence of (AHL)/#3)

- Prof T will now take sample ran in spec and run inside florimidor to check UV absorbance blanking with LB/SOB mixture. Now prof. T. is running the samples in the Fluo. he is blanking w/LB-SOB using 490ex and 510 EM filters Blank read out to .009 +/- .0006 10005 Fluo units for T9002 inside E – Coli Keeping Vol. the same for all spls. blank again before running spls. now running (A) (0) Fluo units we have no florescence (B) (160) Fluo units a little bit of florescence (C) (0) Fluo units this means GFP not producing (D) (0) Fluo units expected (E) (0) Fluo units expected (F) (0) Fluo units expected

Results show no presence of GFP so we will break cells open then test again.

9/28/10

Today we will be picking 2 colonies from the Lacz, LuxR electroporated plates that were streaked on 9/22/10 pg 26 1) we will pick 2 colonies from each plate and transfer 15 mL LB – Lennox 2) and 50 mL LB – Lennox with E – Coli & Agrobacteria growth. ( 2 tubes) 3) poured (2) 40 mL tubes of LB then added 40 mL of Amp to them 4) poured (4) 14 mL tubes of LB then added 14 mL of Amp to them Did all this inside Biological fume hood to keep sterile Now I will make LB – Lennox Broth (1L) 1) Weigh out 10 g LB – Lennox Broth powder and place into 500 mL 18 ohm H2O and dissolve inside microwave using Beverage setting 8 oz then autoclave 2) Repeat (2) twice for (1L) 3) 1) 10.0266 g 2) 10.0698 g 4) Placed each into 1L Capped Elenmyier flasks and added using a 500 mL grad cylinder filling with 500 mL 18 ohm H2O and adding 500 to each 1L flask 5) Capped and microwaved on beverage setting then shook to mix 6) Then placed in autoclave on liquid setting/ 121 degrees C for 15 min will place into fridge for use.

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