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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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