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

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^th 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

Monday 11^th August

ASDF

Resuspended primers in water
• Spin down for 5 seconds
• Inverted several times, set for 3 minutes
• Inverte again, let sit for 3 more minutes
• Invert before use


PCR
• 57uL H2O + 1.6uL dNTP + 16uL buffer + 0.8uL polymerase in tube 1
• 9uL of 1 + 0.25uL BB primer for + 0.25uL primer rev + 0.5uL template (PSBC13) in tube 8
• Add 0.5uL pdCAF3 to tube 1
• 9uL of 1 to tube 2-7, 0.25 rev + 0.25 for to each of the tubes respectively
• Spin down for 5 seconds
• Put tube 2-8 in PCR machine

Tuesday 12^th August

ASDF

456uL H2O, 128uL buffer, 12.8uL dNTP, 6.4uL polymerase into tube A

9uL of A + 0.25uL BB for + 0.25uL BB rev + 0.5uL purified green in tube 8

0.5uL pdCAF to tube A

72uL A to tube 1-6, 9uL A to tube 7, 0.25 BB rev/for to 7, 2uL for/2uL rev to 1-6 respectively

Spin down for 5 seconds

PCR in cycle 1

Tube	Primers	Template	Volume(uL)
1	ndmA 1	pdCAF	80
2	ndmA 2	pdCAF	80
3	ndmB 1	pdCAF	80
4	ndmB 2	pdCAF	80
5	ndmC	pdCAF	80
6	ndmD + gst9	pdCAF	80
7	BB	pdCAF	10
8	BB	Green Optogene	10

Wednesday 13^th August

ASDF

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 14^th August

ASDF

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

Friday 15^th August

ASDF

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 18^th August

ASDF

Inoculate 23 and 24 (PCR) in 5mL LB

Inoculate IG4/IG5 in 5mL LB

Tuesday 19^th August

ASDF

Purification
• Centrifuge 3200g for 10 min
• Discard the culture media
• Add 250uL solution 2/RNAse A, pipet up and down to mix thoroughly
• Transfer suspension into a new 1.5mL microcentrifuge
• Add 250uL solution II, invert until a clear lysate appears
• Add 350uL solution II, then immediately invert several times until a white precipitate forms
• Centrifuge at maximum speed for 10 minutes
• Insert a mini column tube into a 2mL collection tube
• Transfer the cleared supernatant to the column
• Centrifuge at maximum speed for a minute
• Discard the filtrate and reuse the collection tube
• Add 50uL HBC buffer
• Centrifuge at maximum speed for one minute
• Discard the filtrate and reuse collection tube
• Add 700uL DNA wash buffer
• Centrifuge at maximum speed for 1 minute
• Discard the filtrate and reuse the collection tube
• Centrifuge the column for two minutes at maximum speed to dry the column matrix
• Transfer the mini column to a clean 1.5mL microcentrifuge tube
• Add 60uL elution buffer to the column
• Sit at room temp for 1 minute
• Centrifuge at maximum speed for 1 minute
• Store DNA at -20C


Wash cells
• 1mL to fresh tube
• 2.5 minutes at 8000g
• Remove all but the pellet, resuspend the salt
• 2mL salt + 8mL H2O


Make M9
• 500uL 40% glucose
• 100uL MgSO4
• 5uL CM
• 10mL 5x salts
• 1mL casamino acids
• 34mL H2O
• Make 9 test tubes, 3 with IG4, 3 with IG5, and 3 with IG4, each having caffeine, theobromine, or water

Thursday 21^th August

ASDF

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

Monday 25^th August

ASDF

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 26^th August

ASDF

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 27^th August

ASDF

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