Team:UIUC Illinois/Notebook

From 2014.igem.org

PROJECT NOTES
- NOTES BY MONTH

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

Tuesday 22^th July

ASDF

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 23^th July

ASDF

0.166g of EDTA
pH at 8.3

Pro.bb rev
pdCAF3.bb for
Pro pdCAF3 for
BB.pdCAF3 rev

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

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

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

Entire falcon tube was used
Optional 2nd wash with DNA wash was done
DNA is in -20C

Wednessday 23^th July

ASDF

0.166g of EDTA
pH at 8.3

Pro.bb rev
pdCAF3.bb for
Pro pdCAF3 for
BB.pdCAF3 rev

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

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

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

Entire falcon tube was used
Optional 2nd wash with DNA wash was done
DNA is in -20C

Thursday 24^th July

ASDF

25mL 100% ethanol
25mg Erm

Stored in -20C

2mL LB + 20uL BL2 + 0uL Erm
2mL LB + 20uL BL2 + 50uL Erm
2mL LB + 50uL BL2 + 100uL Erm

2mL LB + 20uL DH + 0uL Erm
2mL LB + 20uL DH + 50uL Erm
2mL LB + 20uL DH + 100uL Erm

3 glycerol stocks each strain
500uL glycerol + 500uL strains

Took 1mL of I4/I5 LB cultures into microcentrifuge tubes
Spun for 2 min at 8000 rpm
Removed supernatant
Added 1mL of M9 media, resuspended
Spun for 2 min at 8000 rpm
Removed supernatant
Resuspended in 1mL of M9 media
Total of 4 culture tubes, put in 37C
I4 (50uL) +M9 (5mL) +CM34 (5uL)
I4 (50uL) + M9 (5mL) + Caffeine (500uL) + CM34 (5uL)
I5 (50uL) + M9 (5mL) + CM34 (5uL)
I5 (50uL) + M9 (5mL) + Caffeine (500uL) + CM34 (5uL)

4mL LB + 200uL Erm (1mg/mL)
Inoculate shuttle vector from agar stab
Incubate it on 37C shaker for 2-3 hours
4 gradient test tubes
Incubate overnight at 37C

Final Conc.(ug/mL)	Media(mL)	LB(mL)	Erm(uL)
12.5	0.5	1.5	0
65	0.5	1.5	46.7
80	0.5	1.5	60
100	0.5	1.5	77.8

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

pdCAF3

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

Green
Incubated at 37C waterbath for 1 minute
Gel run at 120V for 25 minutes with a 1kb ladder

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

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

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

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

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

Thursday 7^th August

ASDF

Friday 8^th August

ASDF

20.75uL of 1M K2HPO4
4.25uL of 1M KH2PO4
2.25uL of H2O

5g casamino acids + 50mL H2O

Monday 11^th August

ASDF

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

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

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

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

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

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

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Friday 15^th August

ASDF

4-10 middle colony
17-24 small colony to 50uL LB

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

Tuesday 19^th August

ASDF

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

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

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

1uL cutsmart buffer
1uL Xho
8uL DNA

Monday 25^th August

ASDF

120uL Buffer G
105uL H2O

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

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

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

Tuesday 26^th August

ASDF

Use purification protocol from 8/19

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)

1 plate with 0 CM
1 plate with 5ug/mL CM
2 plates with 10ug/mL CM
2 plates with 20ug/mL CM

5mL LB
IG4/5 inoculations involved 5uL of CM solution (1000x dilution)
Placed in 37C

Wendesday 27^th August

ASDF

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)

	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

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

290mg caffeine + 50mL H2O

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