Team:UIUC Illinois/Notebook

Monday 9^th June

ASDF 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 10^th June

ASDF 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 11^th June

ASDF 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 13^th June

ASDF 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 16^th June

ASDF 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 17^th June

ASDF 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 18^th June

ASDF 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 19^th June

ASDF 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 9^th July

ASDF 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 10^th July

ASDF 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 15^th July

ASDF Retrieved lactobacillus WCFS from Dr. Lu

Wednesday 16^th July

ASDF Incubate 4 tubes with LB+CM

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

Thursday 17^th July

ASDF Made 1mg/mL Erm of 50mL

Used syringe to filter out

25mL Erm into LB agar

Made 13 places

Erm is in -20C

Friday 18^th July

ASDF Streak out Erm-R onto Erm plates (made 2)

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

Monday 21^th July

ASDF 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

Saturday 26^th July

ASDF MRS agar of 200mL

• 3g agar (15g/L)
• 10.41g MRS


300mL LB + 3uL CM34 + IG4

300mL LB + 3uL CM34 + IG5

Monday 28^th July

ASDF 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 30^th July

ASDF Ran gDNA extraction

• Quantification: 1.8 ratio

Thursday 31^th July

ASDF 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 4^st August

ASDF 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

Tuesday 5^th August

ASDF Ran gel of pdCAF3 digestion

Set up feeding experiment cultures

• Added 50uL caffeine M9/theobromine M9 to 250mL flasks
• Added 50uL of CM34 to 4 of 6 flasks
• Added 500uL of cells to flasks
• Took 1mL of each culture as zero time point, placed in -20C
• Placed flasks in 30C shaker

Wednesday 6^th August

ASDF

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 7^th August

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

Friday 8^th August

ASDF

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