iGEM 2014 — Team UIUC_Illinois

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# Team:UIUC Illinois/Notebook

### From 2014.igem.org

[![](https://static.igem.org/mediawiki/2013/1/17/PB_LogoHome.gif)](https://2014.igem.org/Team%3AUIUC_Illinois "Home Page")

* [PROJECT](https://2014.igem.org/Team%3AUIUC_Illinois/Project/PuppyProbiotic "PuppyProbiotic")

  + [PUPPY PROBIOTIC](https://2014.igem.org/Team%3AUIUC_Illinois/Project/PuppyProbiotic "PuppyProbiotic")
  + [FUTURE DIRECTIONS](https://2014.igem.org/Team%3AUIUC_Illinois/Project/Future "Future Directions")
  + [MODELING](https://2014.igem.org/Team%3AUIUC_Illinois/Modeling "Bioreactor")
* [ACHIEVEMENTS](https://2014.igem.org/Team%3AUIUC_Illinois/Results "Achievements")

  + [RESULTS](https://2014.igem.org/Team%3AUIUC_Illinois/Results "Results")+ [PARTS](https://2014.igem.org/Team%3AUIUC_Illinois/Parts "Parts")
    + [MEDAL FULFILLMENT](https://2014.igem.org/Team%3AUIUC_Illinois/Medal_Fulfillment "Medal Fulfillment")
* [SOFTWARE](https://2014.igem.org/Team%3AUIUC_Illinois/Software/Cutsultant "Cutsultant")

  + [CUTSULTANT](https://2014.igem.org/Team%3AUIUC_Illinois/Software/Cutsultant "Cutsultant")
  + [EVOLVABILITY](https://2014.igem.org/Team%3AUIUC_Illinois/Software/Evolvalvability "Evolvability")
* [HUMAN PRACTICE](https://2014.igem.org/Team%3AUIUC_Illinois/Human_Practice/ethics "ethics")

  + [ETHICS](https://2014.igem.org/Team%3AUIUC_Illinois/Human_Practice/ethics "ethics")
  + [OUTREACH](https://2014.igem.org/Team%3AUIUC_Illinois/Human_Practice/Outreach "Outreach")
* [SAFETY](https://2014.igem.org/Team%3AUIUC_Illinois/Safety "Safety")
* [NOTEBOOK](https://2014.igem.org/Team%3AUIUC_Illinois/Notebook "Notebook")

  + [NOTES](https://2014.igem.org/Team%3AUIUC_Illinois/Notebook "Notes")
  + [PROTOCOLS](https://2014.igem.org/Team%3AUIUC_Illinois/Protocols "Protocols")
  + [BIBLIOGRAPHY](https://2014.igem.org/Team%3AUIUC_Illinois/Bibliography "Bibliography")
* [TEAM](https://2014.igem.org/Team%3AUIUC_Illinois/Team "Team")

  + [OUR TEAM](https://2014.igem.org/Team%3AUIUC_Illinois/Team "Team")
  + [OFFICIAL PROFILE](https://igem.org/Team.cgi?year=2014&team_name=UIUC_Illinois "Official")

[![](https://static.igem.org/mediawiki/2013/d/d0/PB_LogoiGEM.gif)](https://2014.igem.org/Main_Page "iGEM 2014")

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* PROJECT NOTES
  + NOTES BY MONTH
    - June
      * [Monday 9th June](#Monday_9th_June.html)
      * [Tuesday 10th June](#Tuesday_10th_June.html)
      * [Wednesday 11th June](#Wednesday_11th_June.html)
      * [Friday 13th June](#Friday_13th_June.html)
      * [Monday 16th June](#Monday_16th_June.html)
      * [Tuesday 17th June](#Tuesday_17th_June.html)
      * [Wednesday 18th June](#Wednesday_18th_June.html)
      * [Thursday 19th June](#Thursday_19th_June.html)
    - July
      * [Wednesday 9th July](#Wednesday_9th_July.html)
      * [Thursday 10th July](#Thursday_10th_July.html)
      * [Tuesday 15th July](#Tuesday_15th_July.html)
      * [Wednesday 16th July](#Wednesday_16th_July.html)
      * [Thursday 17th July](#Thursday_17th_July.html)
      * [Friday 18th July](#Friday_18th_July.html)
      * [Monday 21nd July](#Monday_21st_July.html)
      * [Tuesday 22rd July](#Tuesday_22nd_July.html)
      * [Wednesday 23th July](#Wednesday_23rd_July.html)
      * [Thursday 24th July](#Thursday_24th_July.html)
      * [Saturday 26th July](#Saturday_26th_July.html)
      * [Monday 28th July](#Monday_28th_July.html)
      * [Wednesday 30th July](#Wednesday_30th_July.html)
      * [Thursday 31st July](#Thursday_31st_July.html)
    - August
      * [Monday 4st August](#Monday_4th_August.html)
      * [Tuesday 5nd August](#Tuesday_5th_August.html)
      * [Wednesday 6th August](#Wednesday_6th_August.html)
      * [Thursday 7th August](#Thursday_7th_August.html)
      * [Friday 8th August](#Friday_8th_August.html)
      * [Monday 11th August](#Monday_11th_August.html)
      * [Tuesday 12th August](#Tuesday_12th_August.html)
      * [Wednesday 13th August](#Wednesday_13th_August.html)
      * [Thursday 14th August](#Thursday_14th_August.html)
      * [Friday 15th August](#Friday_15th_August.html)
      * [Monday 18th August](#Monday_18th_August.html)
      * [Tuesday 19th August](#Tuesday_19th_August.html)
      * [Thursday 21th August](#Thursday_21th_August.html)
      * [Monday 25th August](#Monday_25th_August.html)
      * [Tuesday 26th August](#Tuesday_26th_August.html)
      * [Wednesday 27th August](#Wednesday_27th_August.html)
    - September
      * [Tuesday 3rd September](#Tuesday_3rd_September.html)
      * [Wednesday 4th September](#Wednesday_4th_September.html)
      * [Thursday 5th September](#Thursday_5th_September.html)
      * [Friday 6th September](#Friday_6th_September.html)
      * [Saturday 7th September](#Saturday_7th_September.html)
      * [Sunday 8th September](#Sunday_8th_September.html)
      * [Monday 16th September](#Monday_16th_September.html)
      * [Tuesday 17th September](#Tuesday_17th_September.html)
      * [Wednesday 18th September](#Wednesday_18th_September.html)
      * [Sunday 22nd September](#Sunday_22nd_September.html)
      * [Monday 23rd September](#Monday_23rd_September.html)
      * [Tuesday 24th September](#Tuesday_24th_September.html)
      * [Wednesday 25th September](#Wednesday_25th_September.html)
      * [Thursday 26th September](#Thursday_26th_September.html)
      * [Friday 27th September](#Friday_27th_September.html)
      * [Saturday 28th September](#Saturday_28th_September.html)
      * [Monday 30th September](#Monday_30th_September.html)
    - October
      * [Tuesday 1st October](#Tuesday_1st_October.html)
      * [Wednesday 2nd October](#Wednesday_2nd_October.html)
      * [Thursday 3rd October](#Thursday_3rd_October.html)
      * [Monday 14th October](#Monday_14th_October.html)
      * [Tuesday 15th October](#Tuesday_15th_October.html)
      * [Wednesday 16th October](#Wednesday_16th_October.html)
      * [Thursday 17th October](#Thursday_17th_October.html)
      * [Friday 18th October](#Friday_18th_October.html)
      * [Saturday 19th October](#Saturday_19th_October.html)
      * [Tuesday 22nd October](#Tuesday_22nd_October.html)
      * [Thursday 24th October](#Thursday_24th_October.html)
      * [Friday 25th October](#Friday_25th_October.html)
      * [Saturday 26th October](#Saturday_26th_October.html)
      * [Sunday 27th October](#Sunday_27th_October.html)

## Monday 9th June

 [ASDF](https://2014.igem.org/Team%3AUIUC_Illinois/Project/PuppyProbiotic) Made LB broth using 10 grams of bacto agar + 12 grams HiVeg LB broth + 600 mL ddH2O

Autoclaved 1.5 mL Eppendorf tubes as well as LB

Poured plates of LB and chloramphenicol (CAM)

Streaked 2 plates with E. coli DguaB (pDCAF3)

Incubated in 37 degrees celsius

Aliquoted kanamycin (KAN) in four 1mL tubes

## Tuesday 10th June

 [ASDF](https://2014.igem.org/Team%3AUIUC_Illinois/Project/PuppyProbiotic) Autoclaved at 1:30pm

* 0.8 grams of YNB + 156 mL H2O
* 28 grams of salts + 490 mL H2O

Used sterile filter for 1M MgSO4 and 1M CaCl2

Suspended 5 uL of CM34 (antibiotic) + 5 mL of LB + 1 colony of pDCAF3 E. coli

Added .5 grams of caffeine + 400 uL of 1M MgSO4 + 20 uL 1M CaCl2 + 40 mL M95x salts + H2O to YNB + H2O mixture

* Labeled this "M9 media"

Inoculated frozen stock of E. coli guaB pDCAF3 in 5 mL of M9 media

Placed sample in 30 degree celsius shaker for culturing

## Wednesday 11th June

 [ASDF](https://2014.igem.org/Team%3AUIUC_Illinois/Project/PuppyProbiotic) Inoculated CBB1 frozen stock in 5 mL of M9 media

Placed sample in 30 degree celsius shaker

Purified pDCAF plasmid using DNA mini kit

* Plasmid concentration was 132.9 ng/uL

## Friday 13th June

 [ASDF](https://2014.igem.org/Team%3AUIUC_Illinois/Project/PuppyProbiotic) Added 10 uL deionized H2O into the J23114 promoter plasmid (Ampᴿ)

Followed bacterial transformation protocol to prepare/grow culture overnight

Inoculated 5 mL of M9 with CBB1 and placed it in 30 degree celsius shaker for 3 days

* Previous CBB1 culture did not grow, showed slight growth
* pDCAF3 strain in M9 did grow in the 30 degree celsius shaker

## Monday 16th June

 [ASDF](https://2014.igem.org/Team%3AUIUC_Illinois/Project/PuppyProbiotic) No growth in M9 by CBB1

To grow the promoter plasmid:

* 5 mL of LB + 1 uL Amp
* Label tubes
* Grow in 30 degree celsius shaker overnight

New CBB1 trials:

* 2 tubes of 1 mL M9 + 2 tubes of 1 mL LB each with 10 uL of sample

Purify genomic DNA plasmid following the Wizard Protocol

* Added lysis buffer before centrifuging. So we separated (translate) sample into 2 tubes and will (translate) accordingly
* We put our 2 promoter plasmid plates in the 4 degree celsius shelf

## Tuesday 17th June

 [ASDF](https://2014.igem.org/Team%3AUIUC_Illinois/Project/PuppyProbiotic) Quantified gDNA with TECAN

* Result: 5.4 ng/uL

Made 2 plates of M9 culture and 2 plates of CBB1 culture (100 uL used) then placed them in 37 degree celsius shaker

## Wednesday 18th June

 [ASDF](https://2014.igem.org/Team%3AUIUC_Illinois/Project/PuppyProbiotic) Autoclaved 50% glycerol

Made four 25% glycerol stock solutions (stored in the -80 degree celsius freezer):

* CBB1 in M9 (x2 1.5 mL tubes)
* Promoter 1
* Promoter 2 (both promoter plasmids are J23114 Ampᴿ)

Purified both promoter plasmids using the purification protocol

* Stored in the -20 degree celsius freezer in the DNA box

## Thursday 19th June

 [ASDF](https://2014.igem.org/Team%3AUIUC_Illinois/Project/PuppyProbiotic) Quantified promoter plasmid (J23114 #1 and #2)

#1: 342.9 ng/ul with a ratio of 1.9
#2: 373.2 ng/ul with a ratio of 1.91

Checking to see if expired enzymes work: Using the J23114 promoter plasmid, we will see if the 3-year expired SpeI works

* We combined 1 uL of 2.1 buffer, 1 uL of Spe1, and 6 uL of J23114 promoter
* Heat bath at 37 degrees celsius for 1 hour because 2.1 buffer is <100% effective on this enzyme
* Then we will run gel electrophoresis: Concluded that the enzyme works

Cutting the promoter with restriction enzymes

* Combined 24 uL of J23114 promoter + 3 uL of CutSmart buffer + 1 uL of each EcoRI and SpeI
* Heat bath at 37 degrees celsius for 1 hour + 20 minute heat-kill at 80 degrees celsius

Cutting linearized backbone

* Linearized backbone with chloraphenicol marker (12 uL) + 12 uL water + 1 uL of each EcoRI, PstI, and DpnI + 3 uL 2.1 buffer
* Heat bath at 37 degrees celsius for 1 hour + 20 minute heat-kill at 80 degrees celsius

## Wednesday 9th July

 [ASDF](https://2014.igem.org/Team%3AUIUC_Illinois/Project/PuppyProbiotic) Made 400mL of M9 solution

* Made 40% glucose solution of 50mL: 2g glucose
* Added the following to 316mL of DI H2O: Na2HPO4: 13.56g Na2HPO4, 2g NH4Cl, 1g NaCl, 6g KH2PO4
* Added 80mL 5x M9 salt solution (20% of 400mL)
* Added 800uL MgSO4 + 40uL CaCl2
* Added 4mL glucose solution using syringe
* Stirred until all dissolved

Removed competent cells from the -80 freezer

Thawed on ice for 30 minutes

Removed CAM agar plate from 4 degree cold room

Mixed 4uL of pDCAF3 plasmid with 30uL competent cells in microcentrifuge tube, placed on ice for 30 minutes

Placed mixture in water bath (42C) for 45 seconds

Placed mixture on ice for 2 minutes

Added 300uL LB to mixture

Placed in 37 degree shaker for 45 minutes

Plated cells on CAM plate, placed in 37 degree drawer overnight

## Thursday 10th July

 [ASDF](https://2014.igem.org/Team%3AUIUC_Illinois/Project/PuppyProbiotic) GuaB/pDCAF suspension in caffeine/theobromine

* 0.0975g/10mL caffeine, 0.09g/10mL -> 50 mM
* 2.5mL of M9 (with Glucose) + 2.4875mL of H2O + 12.5uL of caffeine/theobromine
* Use syringe to filter out 10mL of caffeine/theobromine into new conical test tubes
* Take out guaB/pDCAF from -80C freezer, suspend it using 10uL pipette tips
* Place them into 37C inclubator overnight

## Tuesday 15th July

 [ASDF](https://2014.igem.org/Team%3AUIUC_Illinois/Project/PuppyProbiotic) Retrieved lactobacillus WCFS from Dr. Lu

## Wednesday 16th July

 [ASDF](https://2014.igem.org/Team%3AUIUC_Illinois/Project/PuppyProbiotic) Incubate 4 tubes with LB+CM

* Red 1 and 2
* Green 1 and 2
* Placed in shaker at 12:20pm

## Thursday 17th July

 [ASDF](https://2014.igem.org/Team%3AUIUC_Illinois/Project/PuppyProbiotic) Made 1mg/mL Erm of 50mL

Used syringe to filter out

25mL Erm into LB agar

Made 13 places

Erm is in -20C

## Friday 18th July

 [ASDF](https://2014.igem.org/Team%3AUIUC_Illinois/Project/PuppyProbiotic) Streak out Erm-R onto Erm plates (made 2)

Put them into incubator (start: 9:35am)

## Monday 21th July

 [ASDF](https://2014.igem.org/Team%3AUIUC_Illinois/Project/PuppyProbiotic) cdh PCR redo:

* Made 4 master mixes: 5uL gDNA CBB1, 45uL DDH2O

Master Mix 1: Regular

* 20uL Buffer G
* 4uL forward primer
* 4uL reverse primer
* 0.4uL Q5 polymerase
* 2uL template
* 9.6uL H2O

Master Mix 2: Forward primer

* 20uL Buffer G
* 8uL forward primer
* 0uL reverse primer
* 0.4uL Q% polymerase
* 2uL template
* 9.6uL H2O

Master Mix 3: Reverse primer

* 20uL Buffer G
* 0uL forward primer
* 8uL reverse primer
* 0.4uL Q5
* 2uL template
* 9.6uL H2O

Master Mix 4: gDNA (10x)

* 20uL Buffer G
* 4uL forward primer
* 4uL reverse primer
* 0.4uL Q5 polymerase
* 2uL template
* 9.6uL H2O

PCR Procedure: Made 4 10uL samples of each master mix
Set up thermocycler with the following settings:

* 98C: 30 min
* 40x cycles that ended up being:
* 98C for 20 minutes
* Gradient temperature for 20 minutes: 44.9C, 50.8C. 58.7C, 65.1C
* 72C for 80 min
* 72C for 5 minutes
* Paused at 4C

Optimized shuttle vector growth environments: From 1 mg/mL concentrate

* 1mL of each concentration into 2 falcon tubes
* Inoculated 3mL LB with cells + Erm stocks
* Also had 0ug/mL as a negative control
* 8 tubes in total

| Concentrations(ug/mL) | H2O(mL) | Erm(mL) |
| --- | --- | --- |
| 50 | 3.8 | 0.2 |
| 25 | 1.5 | 1.5 |
| 10 | 0.5 | 0.5 |

Inoculating Optogene Liquid Cultures:

* Added 10mL liquid LB and 10uL chloramphenicol to each of the four falcon tubes
* Picked colonies off each labeled plate and added to tubes as follows:
  + Plate
    - Green light 1
    - Green light 2
    - Red light sensor 1
    - Red light sensor 2* Incubated all 4 tubes in 37C shaker at 5:15pm

Inoculated E. coli guaB pdCAF (I4) in 3uL LB + 3uL CM

Inoculated Top 10 pdCAF (I5) in 3uL LB + 3uL CM

Placed in 37C shaker at 5:15pm

## Tuesday 22th July

 [ASDF](https://2014.igem.org/Team%3AUIUC_Illinois/Project/PuppyProbiotic) Made 80uL M9 + glucose solution

Previous solution was contaminated
16uL 5x M9 salts
63.2uL H2O
.4g NH4Cl, .2g NaCl, 1.2g KH2PO4, 2.712g Na2HPO4
16uL MgSO4 + uL CaCl2
.8mL 40% glucose solution using syringe
Stir until dissolved

Erm concentration gradient

Inoculated shuttle vector and top 10 cells
Incubated in 37C

|  | Erm | LB |
| --- | --- | --- |
| 100ug/mL | 0.5mL | 4.5mL |
| 50ug/mL | 0.25mL | 4.75mL |
| 25ug/mL | 0.125mL | 4.875mL |
| 0ug/mL | 0mL | 5mL |

LAB Media (WCFS1) - prepare for Top 10

* Added 5mL MRS into 3 test tubes
* Inoculate WCFS
* Incubate in 37C overnight

Followed plasmid purification protocol on red 1 and green 1 plasmid

* 250uL of each was used
* Optional 2nd wash with DNA buffer was done
* Stored in -20C

Made M9 media

* 63.2mL of H2O + .4g NH4Cl + .2g NaCl + 1.2g KH2PO4 + 2.7g Na2HPO4 + 16mL 5x M9 salt/H2O solution + 160uL MgSO4 + 8uL CaCl2 + .8mL 40% glucose (syringe filtering)

## Wednessday 23th July

 [ASDF](https://2014.igem.org/Team%3AUIUC_Illinois/Project/PuppyProbiotic)

50M EDTA solution* 0.166g of EDTA
* pH at 8.3

Plasmid purification of shuttle vector -> 16.6 ng/uL

Primers diluted + converted to 5uL stocks* Pro.bb rev
* pdCAF3.bb for
* Pro pdCAF3 for
* BB.pdCAF3 rev

M9, caffeine, theobromine for I4/I5* Transferred 1mL of I4/I5 LB cultures into microcentrifuge tubes
* Centrifuged, poured out supernatant
* Resuspended in M9 + glucose
* Diluted 50uM caffeine slution and 50uM theobromine solution down to 2000uM solutions, sterile filtered
* Created 6 total culture tubes/placed in 37C shaker
* 5uL I4 + 5uL M9+glucose
* 5uL I4 + 3.75uL M9+glucose+1.25uL 2000 uM caffeine
* 5uL I4 + 3.75uL M9+glucose + 1.25 uL 2000 uM theobromine
* 5uL I5 + 5uL M9+glucose
* 5uL I5 + 3.75 uL M9+glucose + 1.25uL 2000 uM caffeine
* 5uL I5 + 3.75uL M9+glucose + 1.25uL 2000 uM theobromine

Placed 2 tubes in PCR cycler labeled ‘P’ and ‘S’

Tube P* 30uL H2O
* 10uL Phusion HF
* 1uL dNTP
* 2.5uL BB.pDCAF3 rev primer
* 2.5uL pro.pDCAF3 for primer
* 3.5uL pDCAF3 template
* 0.5uL 5x Phusion DNA Polymerase

Tube S - Shuttle vector* 6uL H2O
* 2uL Phusion HF
* 0.2uL dNTP
* 0.5uL pro.BB rev primer
* 0.5uL pDCAF3.BB for primer
* 0.7uL shuttle vector template
* 0.1uL 5x Phusion DNA polymerase

Redid plasmid purification protocol on Red 1 and Green 1* Entire falcon tube was used
* Optional 2nd wash with DNA wash was done
* DNA is in -20C

## Wednessday 23th July

 [ASDF](https://2014.igem.org/Team%3AUIUC_Illinois/Project/PuppyProbiotic)

50M EDTA solution* 0.166g of EDTA
* pH at 8.3

Plasmid purification of shuttle vector -> 16.6 ng/uL

Primers diluted + converted to 5uL stocks* Pro.bb rev
* pdCAF3.bb for
* Pro pdCAF3 for
* BB.pdCAF3 rev

M9, caffeine, theobromine for I4/I5* Transferred 1mL of I4/I5 LB cultures into microcentrifuge tubes
* Centrifuged, poured out supernatant
* Resuspended in M9 + glucose
* Diluted 50uM caffeine slution and 50uM theobromine solution down to 2000uM solutions, sterile filtered
* Created 6 total culture tubes/placed in 37C shaker
* 5uL I4 + 5uL M9+glucose
* 5uL I4 + 3.75uL M9+glucose+1.25uL 2000 uM caffeine
* 5uL I4 + 3.75uL M9+glucose + 1.25 uL 2000 uM theobromine
* 5uL I5 + 5uL M9+glucose
* 5uL I5 + 3.75 uL M9+glucose + 1.25uL 2000 uM caffeine
* 5uL I5 + 3.75uL M9+glucose + 1.25uL 2000 uM theobromine

Placed 2 tubes in PCR cycler labeled ‘P’ and ‘S’

Tube P* 30uL H2O
* 10uL Phusion HF
* 1uL dNTP
* 2.5uL BB.pDCAF3 rev primer
* 2.5uL pro.pDCAF3 for primer
* 3.5uL pDCAF3 template
* 0.5uL 5x Phusion DNA Polymerase

Tube S - Shuttle vector* 6uL H2O
* 2uL Phusion HF
* 0.2uL dNTP
* 0.5uL pro.BB rev primer
* 0.5uL pDCAF3.BB for primer
* 0.7uL shuttle vector template
* 0.1uL 5x Phusion DNA polymerase

Redid plasmid purification protocol on Red 1 and Green 1* Entire falcon tube was used
* Optional 2nd wash with DNA wash was done
* DNA is in -20C

## Monday 28th July

 [ASDF](https://2014.igem.org/Team%3AUIUC_Illinois/Project/PuppyProbiotic) Diluted caffeine and theobromine solutions for HPLC

* 500uM Caffeine (10mL)
* 500uM Theobromine (10mL)
* 100uM Caffeine (10mL)
* 100uM Theobromine (10mL)
* 250uM Caffeine + 250uM Theobromine (10mL)

(page 169 on bottom, can’t read XW’s part)

* Take 1mL of I4/I5 LB cultures and spin for 2 minutes at 8000 rpm
* Removed supernatant
* Added 1mL of OH salt, resuspended
* 2 minutes at 8000 rpm
* Removed supernatant
* Resuspended in 1mL of M9 media

## Wednesday 30th July

 [ASDF](https://2014.igem.org/Team%3AUIUC_Illinois/Project/PuppyProbiotic) Ran gDNA extraction

* Quantification: 1.8 ratio

## Thursday 31th July

 [ASDF](https://2014.igem.org/Team%3AUIUC_Illinois/Project/PuppyProbiotic) gDNA qualtify: 97.5 ng/mL, 1.76 ratio

cdh PCR

* 30uL H20
* 10uL Phusion HF
* 1uL dNTP 10x
* 2.5uL rev.
* 2.5uL for.
* 3.5uL template
* 0.5uL polymerase
* Made 4 10uL samples from master mix
* Gradient: 44.9, 50.8, 58.9, 65.1C
* Cycle 3 was used

Performed restriction digestion of pdCAF3 and green optogene* pdCAF3

+ .5uL Cutsmart buffer
+ .5uL Saci
+ .5uL EcoRI-HF
+ 7.52uL pdCAF3
+ 36.48uL H2O

* Green
.5uL Cutsmart buffer
.5uL NcoI
.5uL NheI
8.4uL Green optogene
35.1 uL H2O* Incubated at 37C waterbath for 1 minute
* Gel run at 120V for 25 minutes with a 1kb ladder

Cultured IG4/IG5 in M9 and no CM* 3mL M9, 300uL Caffeine, for only IG4, 30uL cells
* Placed in 37 degree shaker

## Monday 4th August

 [ASDF](https://2014.igem.org/Team%3AUIUC_Illinois/Project/PuppyProbiotic) M9 Caffeine Media

* 200uM 200mL caffeine
* 50 M9 5x salts
* .5mL MgSO4
* .025mL CaCl2
* 1.25mL 40% glucose

M9-Theobromine Media* 200mL 200uM Theobromine
* 50mL M9 5x salts
* .5mL MgSO4
* .025mL CaCl2
* 1.25mL 40% glucose

pdCAF3 Digestion* 5uL cutsmart buffer
* 1uL PstI
* .5uL XhoI
* 7.52uL pdCAF3
* 35.98uL H2O
* 1 hour 37C incubation
* 20 min 80C heat inactivation

## Wednesday 6th August

 [ASDF](https://2014.igem.org/Team%3AUIUC_Illinois/Project/PuppyProbiotic)

Digsted pdCAF3 using XhoI* 5uL cutsmart buffer
* .5uL XhoI
* 7.52uL pdCAF3
* 36.98uL H2O
* 1 hour 37C incubation
* Ran gel

Took 24 hour time point for feeding experiment, stored in -20

## Thursday 7th August

 [ASDF](https://2014.igem.org/Team%3AUIUC_Illinois/Project/PuppyProbiotic)

Took 48 hour time points for feeding experiments, stored in -20C

## Friday 8th August

 [ASDF](https://2014.igem.org/Team%3AUIUC_Illinois/Project/PuppyProbiotic)

Made 250uL 100uM (7.5pH) KPi buffer* 20.75uL of 1M K2HPO4
* 4.25uL of 1M KH2PO4
* 2.25uL of H2O

Centrifuged, washed experimental cultures of IG4 with KPi buffer

Resuspended in 25uL KPi buffer

Made 10% casamino acid solution* 5g casamino acids + 50mL H2O

## Wednesday 13th August

 [ASDF](https://2014.igem.org/Team%3AUIUC_Illinois/Project/PuppyProbiotic)

PCR* Add 114uL H2O, 32uL buffer, 3.2uL dNTP, 1.6uL polymerase to tube 1
* 72uL of A to tube 10 + 20uL BB for + 2uL BB rev + 0.5uL purified green
* 72uL of A to tube A + 2uL ndmD + gst9 + 2uL for/rev
* Spin for 5 seconds
* Put in PCR machine

Gel Purification* Cut one gel piece as small as possible
* Add 500uL of buffer QG to tube 1-4, 10, 11, incubate at 50C for 10 minutes - invert several times until dissolved
* Load liquid in column, spin 1 minute at full speed
* Discard flow through, add 500uL QG, spin 1 minute at full speed
* Discard flow through, add 750uL DNA wash buffer, spin 1 minute at full speed
* Discard flow through, spin 1 minute at full speed
* Discard flow through, add 40uL H2O, move column to fresh tube, spin 1 minute at full speed, sit for 1 minute

| Tube | ng/uL | H2O(uL) | uL |
| --- | --- | --- | --- |
| ndmA 1 | 55.8 | N/A | 0,448 |
| ndmA 2 | 64 | 14 | 0.55 |
| ndmB 1 | 37.9 | 12 | 0.79 |
| ndmB 2 | 55.4 | 4 | 0.9 |
| ndmC | 41.9 | N/A | 0.6 |
| ndmD/9 | 85.6 | N/A | 0.6 |
| BB | 2.5 | 20 | 0.76 |

## Thursday 14th August

 [ASDF](https://2014.igem.org/Team%3AUIUC_Illinois/Project/PuppyProbiotic)

Ran gel

Transformation* Throw Top 10 cells on ice for 10-20 minutes
* Add 5uL of GG rxn mix then stir it gently on pipette
* Leave cells on ice for 20-30 minutes
* Heat shock at 42C for 5 minutes
* Put 1mL LB into the tube, mix it, then transfer it and leave it in 37C for 1 hour
* Plate 20uL and 200uL on LB+CM

</html>

## Friday 15th August

 [ASDF](https://2014.igem.org/Team%3AUIUC_Illinois/Project/PuppyProbiotic)

Pick 1-8 big colony* 4-10 middle colony
* 17-24 small colony to 50uL LB

Set up 24 10uL PCR reactions* 260uL PCR
* 52mL buffer
* 5.2uL dNTPs
* 0.5uL for ndmB
* 1.5uL rev ndmB
* 2.5uL polymerase
* 180uL H2O

## Monday 18th August

 [ASDF](https://2014.igem.org/Team%3AUIUC_Illinois/Project/PuppyProbiotic)

Inoculate 23 and 24 (PCR) in 5mL LB

Inoculate IG4/IG5 in 5mL LB

## Thursday 21th August

 [ASDF](https://2014.igem.org/Team%3AUIUC_Illinois/Project/PuppyProbiotic)

Digestion* 1uL cutsmart buffer
* 1uL Xho
* 8uL DNA

## Monday 25th August

 [ASDF](https://2014.igem.org/Team%3AUIUC_Illinois/Project/PuppyProbiotic)

240uL* 120uL Buffer G
* 105uL H2O

PCR* Backbone: 80uL x2 (purified green and associated primers)
* cdhA: 40uL (CBB1 with Xba + cdhBA)
* cdhBC: 40uL (CBB1 with Nsi + cdhAB)

Feeding assay* Inoculated Top 10, IG4, and IG5 in LB
* Autoclaved 3 250mL flasks
* Ran gel of iGEM BB PCR

Inoculation* Inoculated E. coli in 3uL LB + 3uL CM
* 37C shaker

## Tuesday 26th August

 [ASDF](https://2014.igem.org/Team%3AUIUC_Illinois/Project/PuppyProbiotic)

Purifying the shuttle vector plasmid (yielded 34.3 ng/mL, 1.83)* Use purification protocol from 8/19

Electroporated plasmid into lactobacillus (shuttle vector)* 2-mm cuvette, 1.7kV, ~5ms, ~200uL competent cells, 20uL plasmid DNA
* Recovered in 1900uL MRS medium, incubated for 2.5 hours at 37C
* Plated on various CM MRS plates at 30C (25uL Lactobacillus shuttle vector cells)

Made CM-MRS plates* 1 plate with 0 CM
* 1 plate with 5ug/mL CM
* 2 plates with 10ug/mL CM
* 2 plates with 20ug/mL CM

Inoculated IG4, IG5, Top 10* 5mL LB
* IG4/5 inoculations involved 5uL of CM solution (1000x dilution)
* Placed in 37C

## Wendesday 27th August

 [ASDF](https://2014.igem.org/Team%3AUIUC_Illinois/Project/PuppyProbiotic)

Prepared the following:* 10mL 1x M9 salts (2mL 5x + 8mL H2O)
* 40mL concentrated M9 + 1% casamino acids
* 50mL 2mM caffeine solution (58mg caffeine + 50mL H2O)
* 50mL 2mM theobromine solution (54mg theobromine + 50mL H2O)

Washed overnight cultures using 1x M9 salts

Prepared feeding assay as follows (in chart)

Removed 500uL of each culture, labeled with date and time 0, in -20C

Cultures placed in 37C

|  | Tube1 | Tube2 | Tube3 | Tube4 | Tube5 | Tube6 | Tube7 | Tube8 | Tube9 |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Conc. M9+CA(mL) | 4 | 4 | 4 | 4 | 4 | 4 | 4 | 4 | 4 |
| 2mM Caff(mL) | N/A | N/A | 1 | N/A | N/A | 1 | N/A | N/A | 1 |
| 2mM Theo(mL) | N/A | 1 | N/A | N/A | 1 | N/A | N/A | 1 | N/A |
| H2O(mL) | 1 | N/A | N/A | 1 | N/A | N/A | 1 | N/A | N/A |
| Cells(50uL) | Top 10 | Top 10 | Top 10 | Top 10 | Top 10 | Top 10 | Top 10 | Top 10 | Top 10 |
| CM34(uL) | N/A | N/A | N/A | 5 | 5 | 5 | 5 | 5 | 5 |

160mL M9 + 1% casamino acids* 100mL H2O
* 20mL 10% casamino acids
* 20uL 1M cach(?)
* 400uL 1M MgSO4
* 40mL 5 M9 salts

10mM caffeine* 290mg caffeine + 50mL H2O

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