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August

Aug 4

Roxanne and Kirsten

DNA purification of the 2 pSB1A3 plasmids, dT and pTet using the gel extraction kit

Ran gel to check concentrations

• pTet 25ng/µL
• pSB1A3-2
• pSB1A3-1
• dT 180 ng/µL, 3318bp
• N-term 25 ng/µL, 507bp
• Lumazine 120 ng/µL, 861bp
• c-term 25 ng/µL, 55bp
• EYFP 878bp

Ligations of:

pTet-EYFP

Lumazine-dT

c-term-dT

Calculations:

EYFP: 3x(878bp [EYFP]/2211bp [pTet])x25ng

=29.78ng

=4µL EYFP:1 µL pTet

1 µL Lumazine: 1 µL dT

1 µL c-term:1 µL dt

	pTet-EYFP	Lumazine-dT	c-term-dT
milliQ water	3.5 µL	6.5 µL	6.5 µL
10x buffer	1 µL	1 µL	1 µL
insert	4 µL	1 µL	1 µL
Vector	1 µL	1 µL	1 µL
Ligase	0.5 µL	0.5 µL	0.5 µL

Megan/Mackenzie

Gel extraction pSBIA3-1 and 2 (plasmid for biobrick parts)

Transformation: Tranformed to DH5alpha pBAD = promoter TetR= inverter. Added 2 micro liters of each ligation to DH5alpha. See July 14th Protocol

Restrictions of pBAD (SpeI/PstI), TetR (XbaI/PstI), PLacI (SpeI, PstI), sRBS (SpeI/ PstI), mRBS (SpeI/PstI). Used PCR tubes. Each tube contains: 5 micro liters MillQ water, 3 micro liters 10x Tango Buffer, 1 micro liter enzyme 1, 1 micro liter enzyme 2, 20 micro liters of DNA. Put all tubes in thermal controller at 37 degrees.

Roxanne,

Gel Extraction of pBAD, TetR, pLacI, sRBS, mRBS

Lengths:

pBAD:2287 bp

TetR: 902 bp

pLacI: 2279

sRBS: 2092 bp

mRBS: 2092 bp

Lane:

1. Ladder
2. TetR
3. pLacI
4. sRBS
5. mRBS
6. pBAD

Aug 5th

Kirsten and Fan

Gel extraction of TetR, pLacI, sRBS, mRBS, pBAD according to Qiagen protocol.

Ran analytic gel

1µL Dye + 5µL DNA

6µL Ladder

Lane:

1. Ladder
2. TetR
3. pLacI
4. sRBS
5. mRBS
6. pBAD
7. pSB1A3-1
8. pSB1A3-2

tetR concentration: 25ng/µL

everything else: 100ngµL

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Aug 6

Roxanne

Set up the following ligations

pSB1A3-2+n-term tag and

pSB1A3-1+c-term tag

• 6.5µL milliQ water
• 1 µL 10x T4 buffer
• 1 µL insert
• 1 µL vector
• 0.5 µL T4 ligase

pSB1A3+lumazine

• 5.5µL milliQ water
• 1 µL 10x T4 buffer
• 2 µL insert
• 1 µL vector
• 0.5 µL T4 ligase

pBAD+tetR inverter

• 2.5µL milliQ water
• 1 µL 10x T4 buffer
• 5 µL insert
• 1 µL vector
• 0.5 µL T4 ligase

mRBS+n-term tag

• 6.5µL milliQ water
• 1 µL 10x T4 buffer
• 1 µL insert
• 1 µL vector
• 0.5 µL T4 ligase

Colony PCR of pTet-EYFP, lumazine-dT, c-term-dT

Picked 5 colonies of each construct, followed protocol of July 23.

E1-E5=EYFP colonies 1-5

E+=EYFP (no pTet)

L1-L5=lumazine-dT colonies 1-5

L+= lumazine (no dT)

C1-C5= c-term-dT colonies 1-5

C+= c-terminal (no dT)

-=no plasmid

Lisza

Mixed 10mM primers for: antisense suffix primer and FP N-term fusion primer

Ashley, Mackenzie, Lisza

Analytical gel of colony PCR’s (from earlier)

Volumes:

• 10 µL DNA
• 2 µL 6x loading
• 10 µL ladder

Lane

1. 1 kb ladder
2. –ve control
3. L1
4. L2
5. L3
6. L4
7. L5
8. L+
9. E1
10. E2
11. E3
12. E4
13. E5
14. E+
15. C1
16. C2
17. C3
18. C4
19. C5
20. C+

Transformation of mRBS+n-term, pBAD+tetR, pSB1A3+lumazine, pSB1A3+c-term, pSB1A3+n-term

Negative control: water+25 µL DNA

Transformed according to iGEM protocol. Plated on ampicillin plates at 100µL and 400 µL volumes and incubated overnight.

PCR of EYFP and CFP according to protocol of July 30th.

Aug 7

The transformations seemed to have all worked, although there are no colonies on plates marked pSBB1A3+N-term and several on the negative control plate. Speculation: tubes were switched or improper labeling.

Solution: Colony PCR

Lane:

1. Ladder (1kb)
2. Negative
3. TetR
4. pBAD+tetR colony 1
5. pBAD+tetR colony 2
6. pBAD+tetR colony 3
7. mRBS
8. mRBS+nterm colony 1
9. mRBS+nterm colony 2
10. mRBS+nterm colony 3
11. pSB1A3
12. c-term colony 1
13. c-term colony 2
14. c-term colony 3
15. N-term colony 1
16. N-term colony 2
17. N-term colony 3
18. Lumazine colony 1
19. Lumazine colony 2
20. Lumazine colony 3

Set up restriction digest of the preperative PCR of EYFP (c-term) and CFP(c-term) fusion proteins

DPNI, XbaI,PstI

DPNI, EcoRI, speI for the n-term proteins

Picked colonies of the transformed cells for pBAD-TetR, pSB1A3 (n-term), pSB1A3 (c-term), pSB1A3 (lumazine) and mRBS-Nterm

3x in ampicillin 5mL tubes.

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Aug 11

Roxanne

Restricted all the minipreps with EcoRI to check the size of the plasmids prior to sending away for sequencing (10µL rxns)

Restricted:

• pSB-(n-term) w/ SpeI, PstI
• pSB-(c-term) w/ EcoRI, XbaI

in 30 µL rxns to prepare for fusion to CFP of EYFP

Setting up ligations of:

• pSB1A3 + EYFP (c-term)
• pSB1A3 + CFP (c-term)
• pSB1A3 + EYFP (n-term)
• pSB1A3 + CFP (n-term)

mmS6 gene was transformed into DH5α by Megan and Fan, set up a few cultures for minipreps.

Aug 13

Set up restrictions of :

• mmS6 (EcoRI, SpeI)
• n-term CFP (EcoRI, SpeI)
• CheZ (EcoRI, SpeI)
• mRBS-N-term (SpeI, PstI)
• c-term-dt (EcoRI, XbaI)

Gel Extractions of:

• mms6
• cheZ

Using Qiagen bench top protocol

Set up ligations of:

• pSB1A3-1 + mmS6
• pSB1A3-1 + riboswitch
• pSB1A3-1 + n-term CFP
• pSB1A3-1 + n-term EYFP
• pSB1A3-2 + c-term CFP
• pSB1A3-2 + c-term EYFP
• mRBS + N-term CFP
• mRBS + N-term EYFP
• CheZ+dT
• mmS6 + dT

Ran a concentration gel of:

• c-term CFP
• c-term EYFP
• N-term CFP
• N-term EYFP
• c-term-dT
• mRBS-N-term

Concentrations:

• Riboswitch= 100ng/µL
• pSB1A3-1= 100ng/µL
• pSB1A3-2= 100ng/µL
• dT= 180ng/µL

Ran 1% agarose gel (with 2 wells taped together)

Lane:

1. 5µL of 1kb ladder
2. 20µL CheZ, 4µL dy

Ran at 110V for 30min

Gel extraction and purification of CheZ and mms6, mmS6 split into two tubes. Following Qiagen kit purification benchtop protocol.

Concentration Gel (1% agarose)

Lane:

1. 1 kb ladder (6µL)
2. N-term EYFP
3. C-N
4. Y-N
5. 4
6. 1
7. mmS6
8. C-term dT
9. mRBS+n-term

Only 9/18 transformations worked

Success:

• mmS6 in pSB1A3
• mmS6 in dT
• riboswitch in pSB1A3
• CheZ in dT
• CFP (no promoter)
• GFP gene
• pSB1A3 + n-term EFYP (1)
• pSB1A3 +n-term EYFP (2)
• fused c-EYFP

Added more ligase and will re-transform the following:

• Fused NEYFP (1)
• Fused NEYFP (2)
• Fused NCFP (1)
• Fused NCFP (2)
• Fused CEYFP
• Fused CCFP
• pSB1A3 +N-CFP (1)
• pSB1A3 +N-CFP (2)
• pSB1A3 +C-CFP (1)
• pSB1A3 +C-EYFP (1)

Transformed all of the above. Picked colonies of successes.

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Aug 15th

The transformations didn’t work! Restart

Picked 3 colonies of cheZ-dT

1 of mmS6+dT (for mini and maxiprep)

3 of fused C-EYFP

3 of pSB1A3-mmS6

3 of riboswitch in pSB1A3

3 of GFP gene

3 of CFP (no promoter)

2 of the riboswitch and all of the N-EYFP in pSB1A3 turned red suggesting that the RFP reporter was religated in.

Will miniprep the “good” cells and restrict with EcoRI to check for size

Glycerol stock.

Aug 18

Megan and Ashley

Pelleted down cells (small samples and large samples) picked from transformations to prep for mini-preps.

Add 750 x2 micro liters of your small sample solution to centrifuge tube, Add 50 mL of large sample to large centrifuge tubes. Balanced the Centrifuge and ran for 2 minutes at 13000 rpm. Dump out Supernatant/ leave pellet. Add more sample, centrifuge, and remove more supernatant. Add remainder of sample, centrifuge, remove supernatant. Left samples in -20 degree freezer.

Aug 19

Kirsten and Mackenzie

Maxiprep of fused C-EYFP and mmS6-dT

See protocol for July 13th.

Aug 20

Fan and Mackenzie

Miniprep for:

• cheZ-dT
• mmS6-dT
• PSB1A3-mmS6
• GFP
• CFP no promo
• Fused C-EYFP
• Riboswitch (2,4,7,8,9)

Restriction digest with EcoRI

• 10µL DNA
• 2 µL 10x buffer tango
• 4 µL EcoRI
• Total volume = 20 µL

37°C for 1 hour.

Need to run agarose for all restriction digested samples and DNA samples

Roxanne, Mackenzie

Running a gel of restricted minipreps

Checking mini prep concentration with UV spec

Lane

1. 1 kb ladder
2. cheZ-dT 1 (78 µg/mL)
3. cheZ-dT 2 (80 µg/mL)
4. cheZ-dT 3 (106 µg/mL)
5. mmS6-dT 1 (82 µg/mL)
6. mmS6-dT 2(91 µg/mL)
7. mmS6-dT 3 (144 µg/mL))
8. pSB-mmS6 1 (125 µg/mL)
9. pSB-mmS6 2(88 µg/mL)
10. pSB-mmS6 3(135 µg/mL)
11. GFP 1(-------)
12. GFP 2 (108 µg/mL))
13. GFP 3(98 µg/mL)
14. CFP no promo 1(----------)
15. CFP no promo 2 (134 µg/mL)
16. Fused C-EYFP 1 (49 µg/mL)
17. Fused C-EYFP 2 (174 µg/mL)
18. Fused-CEYFP 3 (75 µg/mL)
19. 1 kb ladder)

Lane

1. 6 µL 1 kb ladder
2. 6 µL pSB1A3
3. Riboswitch 2 (152 µg/mL)
4. Riboswitch 4 (81 µg/mL)
5. Riboswitch 7 (68 µg/mL)
6. Riboswitch 8 (112 µg/mL)
7. Riboswitch 9 (88 µg/mL)
8. 6 µL 1 kb ladder

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Aug 25

Roxanne

Set up restrictions

1,4,7 are c-term

2,5,8 are n-term

1,2=EYFP

4,5=CFP

7,8=ECFP

• Fusion 1,4,7 restrict with DPN1, EcoRI, SpeI
• Fusion 1,4,7 restrict with DPN1, XbaI, PstI
• Fusion 2,5,8 restrict with DPN1, EcoRI, SpeI
• Fusion 2,5,8, restrict with DPN1, XbaI, PstI
• Riboswitch restrict with SpeI, PstI
• CheZ-dT restrict with XbaI, PstI
• GFP restrict with EcoRI, SpeI

• 4 µL milliQ water
• 2 µL 10x Tango buffer
• 12 µL DNA
• 1 µL E1
• 1 µL E1

Set up maxipreps for:

• Lumazine-dT
• mmS6-dT

Ligations:

• Fusion 1 + (c-term)-dT
• Fusion 4 + (c-term)-dT
• Fusion 7 + (c-term)-dT
• Fusion 1 + pSB1A3-2
• Fusion 4 + pSB1A3-2
• Fusion 7 + pSB1A3-2
• Fusion 2 + mRBS (n-term)
• Fusion 5 + mRBS (n-term)
• Fusion 8 + mRBS (n-term)
• Fusion 2 +pSB1A3-1
• Fusion 5 +pSB1A3-1
• Fusion 8 +pSB1A3-1
• Riboswitch +CheZ-dT
• GFP + dT
• sRBS + lumazine-dT
• sRBS + mmS6-dT

Aug 26

Lisza

Deactivated to reactions @ 65° for 15 min

Roxanne

Set up liquid cultures of:

• mmS6 in pSB1A3
• mmS6-dT
• lumazine in pSB1A3
• lumazin-dT
• c-term in pSB1A3
• c-term-dT
• n-term in pSB1A3
• mRBS-(n-term)
• pTet-EYFP
• pBAD-TetR
• riboswitch in pSB1A3

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Aug 27

Fan

Miniprep of the O/N culture

[DNA] unknown

DNA was eluted from the spin column in 50mL milliQ water

• 20µL of each DNA sample was sent to iGEM (MIT)

Kirsten

Gel extraction of GFP, mmS6-dT,CheZ-dT

Purified riboswitch, sRBS, c-term-dT, N-term-mRBS. Eluted with 50µL of EB

Into -20°C freezer.

10µL ligations of GFP+dT, sRBS+mmS6-dT and riboswitch+cheZ. Left at room temp for 2 hours until transformation.

Alix

Transformations of ligations following iGEM protocol

Aug 31

Roxanne

PCR of CFP, ECFP, EYFP w/ n-term prefix or c-term suffix

20µL Restrictions of:

• pLacI with SpeI,PstI
• pStrong with SpeI,PstI
• Lumazine-dT with XbaI ,PstI

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September

September 3

Roxanne, Ashley, Kirsten

Made 1.5L agar LB media according to lab protocol. Roxanne is autoclaving tomorrow.

Pick 3 lumazine-dT colonies from glycerol stocks to grow overnight

Restriction Digest

Reaction 1

	Ribo-cheZ-dT	GFP-dT	sRBS-mmS6	Mr. Gene mmS6
milliQ water	5 µL	5 µL	5 µL	5 µL
Buffer tango	3 µL	3 µL	3 µL	3 µL
DNA	20 µL	20 µL	20 µL	20 µL
XbaI	1 µL	1 µL	1 µL	1 µL
PstI	1 µL	1 µL	1 µL	1 µL
total	30 µL	30 µL	30 µL	30 µL

Reaction 2

	Ribo-cheZ-dT	GFP-dT	sRBS-mmS6	Mr. Gene mmS6
milliQ water	3 µL	3 µL	3 µL	3 µL
Buffer tango	1 µL	1 µL	1 µL	1 µL
DNA	5 µL	5 µL	5 µL	5 µL
EcoRI	1 µL	1 µL	1 µL	1 µL
total	10 µL	10 µL	10 µL	10 µL

Into HWB at 37°C for 2 hours

Gel Electrophoresis (concentration):

Lane

1. 1kb ladder
2. ribo-cheZ-dT (EcoRI)
3. GFP-dT (EcoRI)
4. sRBS-mmS6-dT (EcoRI)
5. NEYFP (XbaI,PstI)
6. NEYFP (ecoRI, speI)
7. NCFP (ecoRI, speI)
8. NECFP (xbaI,pstI)
9. CCFP(ecoRI,speI)
10. CECFP(ecoRI,speI)
11. CCFP (notI,pstI)
12. NEYFP (ecoRI,speI)
13. CECFP (xbaI)
14. CEYFP(xbaI,pstI)
15. NCFP (xbaI,pstI)
16. CEYFP(xbaI)
17. pLacI
18. pStrong
19. 1kb ladder

5µL sample, 1 µL loading dye (6x) 6 µL ladder

Ran at 100V for 1 hour

Concentrations good!

Alix

Gel extraction of:

sRBS-mmS6-dT

riboswitch-cheZ-dT

GFP-dT

mmS6

Following Qiagen benchtop protocol

September 4

Ashley

Ligation of:

• C-term-dT +C-CFP #2
• C-term-dT +C-ECFP #2
• C-term-dT + C-EYFP #2
• pSB1A3-2+C-CFP#1
• pSB1A3-2+C-ECFP#1
• pSB1A3-2+C-EYFP#1
• mRBS-N-term +N-CFP#1
• mRBS-N-term +N-EYFP#1
• mRBS-N-term +N-ECFP#1
• pSB1A3-1+N-CFP#2
• pSB1A3-1+N-ECFP#2
• pSB1A3-1+N-EYFP#2

Reaction

	Control	All reactions
milliQ water	7.5 µL	4.5 µL
10x buffer T4	1 µL	1 µL
Vector	1 µL	1 µL
Insert	0 µL	3 µL
T4 DNA ligase	0.5 µL	0.5 µL
total	10 µL	10 µL

Restriction Digest

	Control (no enzyme)	Lumazine-dT (1)
milliQ water	2.4 µL	0.4 µL
10x buffer Tango	1.6 µL	1.6 µL
DNA	16 µL	16 µL
XbaI	0 µL	1 µL
PstI	0 µL	1 µL
total	20 µL	20 µL

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September 8

Ashley

Made 1.5L LB agar according to protocol

Made 300mL LB broth according to protocol

Gel Electrophoresis (for extraction)

5 µL ladder

5 µL sample, 1 µL dye

Lane

1. 1 kb ladder
2. empty^
3. empty
4. lumazine-dT (XbaI, PstI) 600bp

^there was a control (no enzyme) but not enough DNA to load

Ran at 1000V for one hour

Gel extraction according to Qiagen benchtop protocol

Sept 10

Ashley, Kirsten

Ran 1% agarose

Lane

1. 1 kb ladder
2. C-term-dT + C-EYFP #2
3. pSB1A3-1+N-CFP#2
4. pSB1A3-1+N-ECFP#2
5. lumazine+sRBS
6. pSB1A3-2+C-EYFP#1
7. pSB1A3-1+N-EYFP#2
8. mRBS-N-term +N-CFP#1
9. mmS6+pSB1A3-1
10. GFP-dT
11. mmS6-dT
12. pSB1A3-2+C-ECFP#1
13. mRBS-N-term +N-ECFP#1
14. mRBS-N-term +N-EYFP#1
15. C-term-dT +C-CFP #2
16. C-term-dT +C-ECFP #2
17. pSB1A3-2+C-CFP#1

3 µL sample + 0.5 µL dye

6 µL 1 kb ladder

Mackenzie

Transformation into DH5α

• sRBS-lumazine-dT (km)
• mmS6-dT (km)
• pSB1A3+mmS6
• mRBS-N-term +N-ECFP#1
• mRBS-N-term +N-EYFP#1
• C-term-dT +C-ECFP #2
• C-term-dT + C-EYFP #2
• pSB1A3-2+C-ECFP#1
• pSB1A3-2+C-EYFP#1

The following were not transformed due to lack of competent cells:

• pSB1A3-1+N-ECFP#2
• pSB1A3-1+N-EYFP#2
• GFP-dT

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September 14

Ashley

PCR of EYFP, CFP and ECFP

See Aug 6 for volumes

CFP(1)=prefix primer and antisense C-term suffix

CFP(2)=N-term prefix and antisense suffix

EYFP(1)=prefix primer and antisense C-term suffix

EYFP(2)=N-term prefix and antisense suffix

ECFP(1)=prefix primer and antisense C-term suffix

ECFP(2)=N-term prefix and antisense suffix

Cycle: tail67

Sept 16

Ashley

Miniprep of mmS6-dT 1,2 and 3. Used Qiagen benchtop protocol and kit. Eluted with 50 µL buffer EB. Put into -20°C freezer orange tray.

PCR Purification of restriction digest of PCR products.

September 24

Ashley, Kirsten

1% gel electrophoresis 6uL ladder (1kb) 5uL sample +1 uL 6x loading dye 100V, 40min

Lane

1. 1kb Ladder
2. C-CFP-1
3. C-ECFP-1
4. N-EYFP-2
5. N-CFP-2
6. N-ECFP-2
7. C-EYFP-1
8. CFP-1 Purified PCR
9. ECFP-1 Purified PCR
10. EYFP-2 Purified PCR
11. CFP-2 Purified PCR
12. ECFP-1 Purified PCR
13. EYFP-1 Purified PCR
14. C-CFP(1)
15. ECFP(1)
16. C-EYFP(1)
17. N-CFP(2)
18. N-EYCFP(2)
19. N-EYFP(2)
20. 1KB LADDER

Gel was run to check for DNAses, found that DPN-1 had DNAses.

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Sept 25

1% agarose gel 100V, 40min

Lane:

1. 1kb ladder
2. mms6(EcoRI,SpeI)
3. pLacI (SpeI, PstI)
4. Lumazine-dT

Only pLacI success, gell extracted and eluted with 50uL buffer EB.

Miniprep of:

• 3x mmS6-dT
• 3x pSB1A3-mmS6
• 2x cheZ-dT
• 1x riboswitch-cheZ-dT
• 2x GFP-dT
• 3x C-EYFP-dT

Restricted all of the above with EcoRI

• 8uL DNA
• 1uL 10x buffer tango
• 1uL EcoRI

for 1 hour.

Restriction of fusion proteins with either EcoRI,SpeI or XbaI,PstI

• Heat inactivated a 60C for 10min

Ran on a gel (1% agarose)100V,40min

Lane:

1. 1kb ladder
2. pSB-mmS6 #1
3. pSB-mmS6 #2
4. pSB-mmS6 #3
5. mms6-dT #1
6. mms6-dT #2
7. mms6-dT #3
8. EYFP C-term-dT #1
9. EYFP C-term-dT #2
10. EYFP C-term-dT #3
11. GFP-dT#1
12. GFP-dT#2
13. Riboswitch-cheZ-dT
14. cheZ-dT#1
15. cheZ-dT#2
16. C-CFP (EcoRI,SpeI)
17. C-CFP (XbaI,PstI)
18. C-ECFP (EcoRI,SpeI)
19. C-ECFP (XbaI,PstI)
20. 1kb laddder

• Restriction of Fusion proteins with Dpn1
• Heat inactivated a 60C for 10min

Lane:

1. 1kB ladder
2. N-ECFP
3. N-ECFP
4. N-EYFP
5. E-EYFP
6. N-CFP
7. N-CFP
8. C-EYFP
9. C-EYFP
10. 1kB ladder

Restriction of Lumazine-dT

• 30uL DNA
• 4uL 10x buffer
• 3uL EcoRI
• 3uL SpeI

Setup Liquid culture of the Mr. Gene mmS6 x3

September 28

Ashley

1% agarose gel

• 5uL DNA+1uL 6x loading dye
• 6uL ladder (1kb)

Lane:

1. 1kb ladder
2. N-ECFP (XbaI,PstI)
3. N-ECFP (EcoRI, SpeI)
4. N-EYFP(XbaI,PstI)
5. N-EYFP(EcoRI, SpeI)
6. N-CFP(XbaI,PstI)
7. N-CFP(EcoRI, SpeI)
8. C-EYFP(XbaI,PstI)
9. C-EYFP(EcoRI, SpeI)
10. 1kb ladder

Ran at 100V for 40 min

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October

October 1

Roxanne Gel for gel extraction

Restriction Ribo-Chez-dT

• 2x CheZ-dT
• 2x GFP-dT
• 3x C-EYFP-dT

Ligation

• C-ECFP-dT
• C-CFP-dT
• C-EYFP-dT
• mRBS-N-ECFP
• mRBS-N-EYFP
• pSB-C-EFP
• pSB-C-EYFP
• pSB-C-CFP
• pSB-N-ECFP
• pSB-N-EYFP

Aliquoted DH5α cells

Ashley

Tube	Tube + Gel	Gel
1.1226g	1.4931g	0.2671g
1.012g	1.2302g	0.1290g

Eluted with 50uL buffer EB

Ligation

	1:3	2:6
lum-dT	3uL	6uL
SRBS	1uL	2uL
Ligase Buffer	1uL	2uL
Water	4.5uL	9uL
Ligase	0.5uL	1uL

Oct 2

Pick Colonies:

• sRBS + Lumazine-dT
• mRBS + N-term FPs
• C-term FPs + dT
• pSB + N-term FPs
• pSB + N-term FPs

Gel Extraction:

• Riboswitch-cheZ-dT
• 2x cheZ-dT (in case Ribo-cheZ-dT isn't actually there)
• 3x C-EYFP-dT
• 2x GFP-dT

Liquid Culture:

• Riboswitch-cheZ-dT
• 2x cheZ-dT (in case Ribo-cheZ-dT isn't actually there)
• 3x C-EYFP-dT
• 2x GFP-dT

Miniprep:

• Mr. Gene mms6

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Sat Oct 3

Miniprep:

• sRBS + Lumazine-dT
• mRBS + N-term FPs
• C-term FPs + dT
• pSB + N-term FPs
• pSB + N-term FPs
• sRBS + Lumazine-dT

Restriction:

Analytic and Prep:

• mRBS + N-term FPs
• C-term FPs + dT
• pSB + N-term FPs
• pSB + N-term FPs

Analytic only:

• Mr. Gene mms6

Prep only:

• pBAD-TetR

Oct 5

Gel extraction:

• sRBS + Lumazine-dT
• mRBS + N-term FPs
• C-term FPs + dT
• pSB + N-term FPs
• pSB + N-term FPs

Ligation:

• pStrong + Riboswitch-cheZ-dT
• Riboswitch-GFP-dT
• pLac + sRBS-Lumazine-dT
• pBad-TetR + C-term FPs
• pBad-TetR + N-term FPs
• pTetR + N-term

Transformation:

• pLac + sRBS-Lumazine-dT
• pBad-TetR + C-term FPs
• pBad-TetR + N-term FPs
• pTetR + N-term ECFP
• pStrong + Riboswitch-cheZ-dT
• Riboswitch-GFP-dT

Sequencing:

• sRBS + Lumazine-dT
• mRBS + N-term FPs
• C-term FPs + dT
• pSB + N-term FPs
• pSB + N-term FPs
• Mr. Gene mms6

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Tues Oct 6

Pick Colonies:

• pLac + sRBS-Lumazine-dT
• pBad-TetR + C-term FPs
• pBad-TetR + N-term FPs
• pTetR + N-term ECFP
• pStrong + Riboswitch-cheZ-dT
• Riboswitch-GFP-dT

Wed Oct 7

Miniprep:

• pLac + sRBS-Lumazine-dT
• pBad-TetR + C-term FPs
• pBad-TetR + N-term FPs
• pTetR + C-term ECFP
• pTetR + N-term ECFP
• pStrong + Riboswitch-cheZ-dT
• Riboswitch-GFP-dT

Analytic Restriction:

• pLac + sRBS-Lumazine-dT
• pBad-TetR + C-term FPs
• pBad-TetR + N-term FPs
• pTetR + N-term ECFP
• pStrong + Riboswitch-cheZ-dT
• Riboswitch-GFP-dT

Prep Restriction:

• Riboswitch-GFP-dT

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Thurs Oct 8

Gel Extraction:

• Riboswitch-GFP-dT

Ligation:

• pStrong + Riboswitch-GFP-dT

Transformation:

• pStrong + Riboswitch-GFP-dT

Sequencing:

• pLac-sRBS-Lumazine-dT
• pBad-TetR-C-term FPs
• pBad-TetR-N-term FPs
• pTetR-N-term ECFP
• pStrong-Riboswitch-cheZ-dT
• Riboswitch-GFP-dT

Maxiprep:

• pLac-sRBS-Lumazine-dT
• pBad-TetR-C-term FPs
• pBad-TetR-N-term FPs
• pTetR-N-term ECFP
• pStrong-Riboswitch-GFP-dT

Restriction:

• pLac-sRBS-Lumazine-

Analytic and Prep:

• pBad-TetR-C-term FPs
• pBad-TetR-N-term FPs
• pTetR-N-term ECFP
• C-term CFP (has pTetR)

Analytic Only:

• pStrong-Riboswitch-GFP-dT

Fri Oct 9 Overexpression pLac + sRBS-Lumazine-dT Gel Extraction pLac-sRBS-Lumazine-dT pBad-TetR-C-term FPs pBad-TetR-N-term FPs pTetR-N-term ECFP C-term CFP (has pTetR) Motility Media

Oct 12

Ligation:

• pLac-sRBS-Lumazine-dT + pBad-TetR-C-term EYFP
• pLac-sRBS-Lumazine-dT + pBad-TetR-N-term EYFP

Transformation:

• pLac-sRBS-Lumazine-dT + pBad-TetR-C-term EYFP
• pLac-sRBS-Lumazine-dT + pBad-TetR-N-term EYFP

Plate Testing:

• pStrong-Riboswitch-GFP-dT

Motility Test:

• pStrong + Riboswitch-cheZ-dT

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Oct 13

Pick Colonies

• pLac-sRBS-Lumazine-dT + pBad-TetR-C-term EYFP
• pLac-sRBS-Lumazine-dT + pBad-TetR-N-term EYFP

Analysis of Plate and Motility Tests

Wed Oct 14

Miniprep:

• pLac-sRBS-Lumazine-dT-pBad-TetR-C-term EYFP
• pLac-sRBS-Lumazine-dT-pBad-TetR-N-term EYFP

Restriction:

• pLac-sRBS-Lumazine-dT-pBad-TetR-C-term EYFP
• pLac-sRBS-Lumazine-dT-pBad-TetR-N-term EYFP

Thurs Oct 15

Gel Extraction:

• pLac-sRBS-Lumazine-dT-pBad-TetR-C-term EYFP
• pLac-sRBS-Lumazine-dT-pBad-TetR-N-term EYFP

Ligation:

• pLac-sRBS-Lumazine-dT-pBad-TetR-C-term EYFP + C-term CFP
• pLac-sRBS-Lumazine-dT-pBad-TetR-N-term EYFP + pTetR-N-term CFP

Transformation:

• pLac-sRBS-Lumazine-dT-pBad-TetR-C-term EYFP + C-term CFP
• pLac-sRBS-Lumazine-dT-pBad-TetR-N-term EYFP + pTetR-N-term CFP

Expression Testing:

• pLac-sRBS-Lumazine-dT-pBad-TetR-C-term EYFP
• pLac-sRBS-Lumazine-dT-pBad-TetR-N-term EYFP

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Fri Oct 16

Pick Colonies:

• pLac-sRBS-Lumazine-dT-pBad-TetR-C-term EYFP + C-term CFP
• pLac-sRBS-Lumazine-dT-pBad-TetR-N-term EYFP + pTetR-N-term CFP

Sat Oct 17

Maxiprep:

• pLac-sRBS-Lumazine-dT-pBad-TetR-C-term EYFP + C-term CFP
• pLac-sRBS-Lumazine-dT-pBad-TetR-N-term EYFP + pTetR-N-term CFP

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