Team:Lethbridge/Notebook

From 2009.igem.org

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Revision as of 19:01, 21 October 2009

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:

Ladder
TetR
pLacI
sRBS
mRBS
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:

Ladder
TetR
pLacI
sRBS
mRBS
pBAD
pSB1A3-1
pSB1A3-2

tetR concentration: 25ng/µL

everything else: 100ngµL

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 kb ladder
–ve control
L1
L2
L3
L4
L5
L+
E1
E2
E3
E4
E5
E+
C1
C2
C3
C4
C5
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:

Ladder (1kb)
Negative
TetR
pBAD+tetR colony 1
pBAD+tetR colony 2
pBAD+tetR colony 3
mRBS
mRBS+nterm colony 1
mRBS+nterm colony 2
mRBS+nterm colony 3
pSB1A3
c-term colony 1
c-term colony 2
c-term colony 3
N-term colony 1
N-term colony 2
N-term colony 3
Lumazine colony 1
Lumazine colony 2
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.

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:

5µL of 1kb ladder
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 kb ladder (6µL)
N-term EYFP
C-N
Y-N
4
1
mmS6
C-term dT
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.

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 kb ladder
cheZ-dT 1 (78 µg/mL)
cheZ-dT 2 (80 µg/mL)
cheZ-dT 3 (106 µg/mL)
mmS6-dT 1 (82 µg/mL)
mmS6-dT 2(91 µg/mL)
mmS6-dT 3 (144 µg/mL))
pSB-mmS6 1 (125 µg/mL)
pSB-mmS6 2(88 µg/mL)
pSB-mmS6 3(135 µg/mL)
GFP 1(-------)
GFP 2 (108 µg/mL))
GFP 3(98 µg/mL)
CFP no promo 1(----------)
CFP no promo 2 (134 µg/mL)
Fused C-EYFP 1 (49 µg/mL)
Fused C-EYFP 2 (174 µg/mL)
Fused-CEYFP 3 (75 µg/mL)
1 kb ladder)

Lane

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

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

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

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

1kb ladder
ribo-cheZ-dT (EcoRI)
GFP-dT (EcoRI)
sRBS-mmS6-dT (EcoRI)
NEYFP (XbaI,PstI)
NEYFP (ecoRI, speI)
NCFP (ecoRI, speI)
NECFP (xbaI,pstI)
CCFP(ecoRI,speI)
CECFP(ecoRI,speI)
CCFP (notI,pstI)
NEYFP (ecoRI,speI)
CECFP (xbaI)
CEYFP(xbaI,pstI)
NCFP (xbaI,pstI)
CEYFP(xbaI)
pLacI
pStrong
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

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 kb ladder
empty^
empty
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 kb ladder
C-term-dT + C-EYFP #2
pSB1A3-1+N-CFP#2
pSB1A3-1+N-ECFP#2
lumazine+sRBS
pSB1A3-2+C-EYFP#1
pSB1A3-1+N-EYFP#2
mRBS-N-term +N-CFP#1
mmS6+pSB1A3-1
GFP-dT
mmS6-dT
pSB1A3-2+C-ECFP#1
mRBS-N-term +N-ECFP#1
mRBS-N-term +N-EYFP#1
C-term-dT +C-CFP #2
C-term-dT +C-ECFP #2
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

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

1kb Ladder
C-CFP-1
C-ECFP-1
N-EYFP-2
N-CFP-2
N-ECFP-2
C-EYFP-1
CFP-1 Purified PCR
ECFP-1 Purified PCR
EYFP-2 Purified PCR
CFP-2 Purified PCR
ECFP-1 Purified PCR
EYFP-1 Purified PCR
C-CFP(1)
ECFP(1)
C-EYFP(1)
N-CFP(2)
N-EYCFP(2)
N-EYFP(2)
1KB LADDER

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

Sept 25

1% agarose gel 100V, 40min

Lane:

1kb ladder
mms6(EcoRI,SpeI)
pLacI (SpeI, PstI)
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:

1kb ladder
pSB-mmS6 #1
pSB-mmS6 #2
pSB-mmS6 #3
mms6-dT #1
mms6-dT #2
mms6-dT #3
EYFP C-term-dT #1
EYFP C-term-dT #2
EYFP C-term-dT #3
GFP-dT#1
GFP-dT#2
Riboswitch-cheZ-dT
cheZ-dT#1
cheZ-dT#2
C-CFP (EcoRI,SpeI)
C-CFP (XbaI,PstI)
C-ECFP (EcoRI,SpeI)
C-ECFP (XbaI,PstI)
1kb laddder

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

Lane:

1kB ladder
N-ECFP
N-ECFP
N-EYFP
E-EYFP
N-CFP
N-CFP
C-EYFP
C-EYFP
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:

1kb ladder
N-ECFP (XbaI,PstI)
N-ECFP (EcoRI, SpeI)
N-EYFP(XbaI,PstI)
N-EYFP(EcoRI, SpeI)
N-CFP(XbaI,PstI)
N-CFP(EcoRI, SpeI)
C-EYFP(XbaI,PstI)
C-EYFP(EcoRI, SpeI)
1kb ladder

Ran at 100V for 40 min

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

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

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

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

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

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

@@ Line 298: / Line 298: @@
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-	<li><a href="https://2009.igem.org/Team:Lethbridge">Home</a></li>
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-===July 3===
-*Ran a 1% agarose gel at 120V for 60 minutes.
-*2uL DNA samples with 2uL 6x loading dye
-Lane 2: 10uL of 2kb ladder,
-Lane 4:undiluted pTet
-Lane 5: undiluted CFP
-Lane 6:undiluted EYFP
-Lane 8: 1/10pTet
-Lane 9: 1/10 CFP
-Lane 10: 1/10 EYFP
-Lane 12: 1/100 pTet
-Lane 13: 1/100 CFP
-Lane 14: 1/100 EYFP
-Lane 16: 1/1000pTet
-Lane 17: 1/1000 CFP
-Lane 18: 1/1000 EYFP
-[[Image:July_3.png|250px]]
-===July 6th===
-'''Jeff, Mackenzie, Ashley'''
-Using 1/10 and 1/100 dilutions of pTET and EYFP to perform large scale digests.
-*pTET:PstI and SpeI
-*EYFP:XbaI and PstI
-reactions each:
-*single digest (PstI/SpeI and XbaI/PstI)
-*double digest
-*no digest
-µL each, means we need 400µL total for each.
-/10 dilution=40µL DNA, 360µL water
-/100 dilution=4µL DNA, 396µL water
-Reaction set up:
-*88µL DNA
-*10µL buffer Tango (10x)
-*1µL RE1 (or water)
-*1µL RE2 (or water)
-*=100 µL total
-reactions run overnight @ 37° C
-'''Kirsten, Lisza'''
-TetR Q04400-plate 1 well 16p-pSB2K3
-LacI promoter-R0010-plate 1 well 1d-Amp
-pBAD-I13453-plate 1, well 1n-pSB1A2
-strong RBS-B0030-plate 1 well 1h-pSB1A2
-med RBS-B0030-plate 1 well 2I-pSB1A2
-µL water into wells
-Transformation:
-µL DNA (TetR, LacI, pBAD, med RBS, strong RBS) into 25µL competent DH5α cells (from the -80 fridge).
-Pipet up then down and swirl. One ice for 30min, then heat shock at 42° C for 45 sec. Ice for 1 min, add 250µL LB broth (from fridge) and incubate in shaker for 1 hour.  Plate onto ampicillin plates (pBAD, LacI, strond and med RBS) and kanamycin plate (TetR) into incubator overnight.  Control: 2.5µL water and 2.5µL DH5α.
-===July 8===
-'''Jeff'''
-Overnight cultures (200mL + antibiotics) of:
-*PlacI (Amp)
-*TetR (Kan)
-*Med RBS (Amp)
-Centrifuged at 5000g, 10 min, 4° for maxiprep
-µL each of each culture used to make glycerol stocks – duplicates made, stored in -80° freezer after flash freezing with liquid nitrogen.
-EYFP insert from AGE separation (incubated in 600µL buffer QG overnight):
-*+200µL isopropanol
-*Incubated on a QiaQuick column
-*13000rpm, 1 min
-Flow through replaced in original tube.  500µL QG applied to column, 1 min centrifugation. Colum washed w/ 750µL PE buffer (1 min centrifuge).  Flow through discarded.  1 more min of centrifugation to get rid of buffer PE.  DNA eluted w/ 25µL water (37°C for 10 min) also a second water elution w/ 25µL water (E2).
-/10 double digest of pTet heat incubated at 85°C for 10 min.
-Maxipreps: 3mL ALSI added, cells responded.  500µL lysozyme added.  15 min @RT,  6mL ALSII added. 10 min at RT.  4.5mL cold ALSIII added, 10 min on ice.  Left a 4°C
-===July 13===
-Setting up ligations:
-µL ligase (stock)
-x T4 DNA ligase buffer
-/10 double digested pTet est 20ng/µL
-E1 (60% yield) = ~5ng/µL; ~1/3 insert size/pTet
-Control needed:
-*1/10 double digested pTet, no ligase
-*1/10 double digested pTet + ligase
-*1/10 double digested pTet + 3x vol of insert
-*1/10 double digested pTet +6x vol of insert
-Reaction 1:
-*1µL T4 DNA ligase 10x buffer
-*1µL double digest pTet
-*8µL water
-Reaction 2:
-*1µL T4 DNA ligase 10x buffer
-*1µL double digest pTet
-*0.5 µL T4 ligase
-*7.5µL water
-Reaction 3:
-*1µL T4 DNA ligase 10x buffer
-*1µL double digest pTet
-*0.5 µL T4 ligase
-*3µL E1 EYFP insert
-*4.5µL water
-Reaction 4:
-*1µL T4 DNA ligase 10x buffer
-*1µL double digest pTet
-*0.5 µL T4 ligase
-*6 µL E1 EYFP insert
-*1.5µL water
-Incubated at 37°C overnight
-Kirsten, Mackenzie
-Chloroform extraction of medRBS, pLacI, TetR, that Jeff started.
-pBab & strong RBS cloudy and phenol chloro done twice.
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-	<li><a href="https://2009.igem.org/Team:Lethbridge">Home</a></li>
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-===July 14===
-'''Megan, Mackenzie, Alix'''
-Transformations of ligation reactions
-Took 2µL of DNA from 1, 2, 3, 4 and added to 25 µL of DH5α cells. Incubated on ice for 30 min. Heat shocked at 42°C for 45 sec. Back to ice for 1 min. For ligations add 500µL of LB broth to each tube, working sterile using a Bunsen burner.  Incubate in shaker at 37°C for1 hour at 200 rpm. Made up two plates (Amp) of each, 100µL and 400µL.
-===July 16th===
-'''Ashley, Mackenzie, Roxanne'''
-Pelleted pTet-EYFP ligation reactions 3 and 4 using at 13000rpm for 1 min.
-Mini-prep of pTet-EYFP ligation reactions 3 and 4 using Qiagen kit + protocol.
-===July 20th===
-'''Lisza'''
-Our genes: added miiliQ water
-*10µL to N-term Arg tail, concentration = 200ng/µL
-*10µL to c-term Arg tail, concentration= 200ng/µL
-*20 µL to lumazine, concentration= 250ng/µL
-===July 21st===
-'''Lisza'''
-Made glycerol stocks of the 4 pTet-EYFP ligations
-'''Kirsten, Alix'''
-Mini-prep of pTet-EYFP (3 &4)x2 followed protocol according to Qiagen.
-Analytic restriction digest of above mini-preps
-Control used mini prepped pTet from June 24.
-Added (in order) to 1.5mL micro centrifuge tubes:
-*12µL water
-*2 µL buffer tango
-*5 µL DNA
-*0.5µL XbaI
-*Total: 20 µL
-'''Megan, Kirsten, Alix, Lisza'''
-Made an 80mL agarose gel for large rig
-.8g agarose
-mL 1x TAE
-Lanes
-#Ladder 1Kb
-#Blank
-#pTet unrestricted
-#pTet undigested
-#rxn III-1 undigested (pTet-EYFP)
-#rxn III-1 digest
-#rxn III-2 undigested
-#rxn III-2 digest (XbaI)
-#blank
-#rxn IV-1 undigested
-#rxn IV-1 digest (XbaI)
-#rxn IV-2 undigested
-#rxn IV-2 digest (XbaI
-Ran at 120V for 40 min( in @ 5:45) stained in ethidium bromide for 10 20 min.
-Picture:
-[[Image:July21pTetEYFP.jpg|250px]]
-'''Lisza'''
-Made  glycerol stocks of maxiprep cultures:
-lumazine
-Arg N-term
-Arg c-term
-Spun down cultures
-Lumazine-1=1.35g
-Lumzine-2= 1.31g
-Lumazine-3=1.29g
-Arg c-term=1.26g
-Arg n-term-1=1.43g
-Arg n-term-2=1.12g
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-</head>
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-	<li><a href="https://2009.igem.org/Team:Lethbridge">Home</a></li>
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-===July 23===
-Did a colony screen PCR:
-colonies from July 14th DH5α Amp
-AB+MC+MT plates labeled 10-32
-With picked colonies transferred bacteria into a 1.5mL tube with 300µL milliQ water. With same toothpick inoculated 5mL (Amp 5mL) grown overnight at 37°C.  Put 1.5L tubes onto heat block at 95°C for 7 min. Centrifuged at full speed for 1 min. Used 5µL supernatant for PCR survey.
-Reactions: control with EYFP
-*10.4µL water (14.9 µL control)
-*2 µL Taq buffer
-*0.4 µL dNTPs
-*1 µL VR primer
-*1 µL VFZ primer
-*0.2 µL Taq polymerase
-*5 µL template (0.5 µL control)
-Cycle: Colony58
-Note: tube 11 contained both samples 11 and 12
-===July 24th===
-% agarose gel at 100V of colony PCR
-Lanes:
-#1 kb Ladder (8 µL)
-#Control
-#Colony 10 (10 µL sample+2 µL dye)
-#Colony 11/12
-#Colony 13
-#Colony 14
-#Colony 15
-#Colony 16
-#Colony 17
-#Colony 18
-#Colony 19
-#Colony 20
-#Colony 21
-#Colony 22
-#Colony 23
-#Colony 24
-#Colony 25
-#Colony 26
-#Colony 27
-#Colony 28
-#Colony 29
-#Colony 30
-#Colony 31
-#Colony 32
-#Colony 33
-#1 Kb ladder (8 µL)
-[[Image:July_24_colony_screen.png|250px]]
-PCR was successful, but had negative colonies.  No pTet EYFP biobrick, only pTet was in the plasmid
-===July 27===
-'''Fan & Lisza'''
-Preparative restriction digest of pTet-EYFP:
-*50µL reactions
-*40 µL DNA
-*5 µLbuffer
-*0.5 µL RE1
-*0.5 µL RE@
-*4 µL milliQ water
-pTet:
-*RE1=pstI
-*RE2=speI
-EYFP:
-*RE1=pstI
-*RE2=xbaI
-Incubated at 37°C for 2 hours
-'''Kirsten, Lisza, Mackenzie'''
-Purified double digested pTet using QiaQuick PCR purification kit. First run through eluted DNA with 50 µL water then done again and eluted with 30 µL  elution buffer. Into -20°C fridge
-Gel Extraction of EYFP
-Weight of tube: 1.0973g
-Weight of tube+gel: 1.9759g
-Weight gel: 0.8786
-Transferred to falcon tube
-Preformed gel extraction according to Qiaquick Gel Extraction kit.  Eluted with 30 µL  of elution buffer.
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-</head>
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-<ul id="qm0" class="qmmc">
-	<li><a href="https://2009.igem.org/Team:Lethbridge">Home</a></li>
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-===July 28===
-'''Kirsten, Alix, Lisza'''
-Ligation of pTet and EYFP
-Reaction 1
-*1.55µL of water
-*2.5 µL 10x buffer
-*5 µL vector (pTet)
-*14.7 µL insert (EYFP)
-*1.25 µLT4 DNA ligase
-*25 µL total
-Reaction 2 (Control)
-*16.25µL of water
-*2.5 µL 10x buffer
-*5 µL vector (pTet)
-*0.0µL insert (EYFP)
-*1.25 µLT4 DNA ligase
-*25 µL total
-Incubate at room temperature for 2 hours then transform
-Restriction Digest of Lumazine 1&2, C-terminal Arg tag, N-terminal Arg tag 1&2.
-*14.6µL of water
-*2.5 µL 10x buffer tango
-*4 µL DNA
-*0.4µ EcoRI
-*21 µL total
-Incubate at 37°C for 2 hours in water bath.
-===July 28===
-'''Fan'''
-Did a transformation with ligation reactions plus a water control.
-===July 29===
-Running gel of the PCR for EYFP and CFP with c-terminal fusion tags and the restriction digests of n-terminal, c-terminal ARG tags and the lumazine gene.
-Loading 6µL ladder, 5µL sample+1µL dye
-Lane:
-#1kb DNA ladder
-#CFP amplicon
-#EYFP amplicon
-#Empty
-#N-terminal #1 (EcoRI digestion)
-#N-terminal #2 (EcoRI digestion)
-#C-terminal (EcoRI digestion)
-#Lumazine # 1 (EcoRI digestion)
-#Lumazine # 2 (EcoRI digestion)
-[[Image:July29.png|250px]]
-Did colony PCR of the 5 colonies from the EYFP-1 plater, 5 of the EYFP-2 plate, 4 of the pTet-1, 4 of the pTet-2, one negative control and one positive (EYFP plasmid). Followed protocol of July 23.
-'''Megan, Lisza, McKenzie'''
-Purify maxipreps using kit. Finished 60 µL of PCR samples with a 5 buffer to 1 PCR sample ratio. Buffer amount = 300 µL. Followed protocol in kit.
-Ran a gel with ladder, 3 wells for PCR, 3 double wells for extraction
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-</head>
-<body>
-<ul id="qm0" class="qmmc">
-	<li><a href="https://2009.igem.org/Team:Lethbridge">Home</a></li>
-	<li><a href="https://2009.igem.org/Team:Lethbridge/Notebook">Top of Page</a></li>
-<li class="qmclear">&nbsp;</li></ul>
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-</body>
-</html>
-===July 30===
-'''Lisza'''
-PCR for fusion c-terminal suffix
-Control: no template DNA
-*1.4 µL milliQ water
-*4 µL buffer (5x Phusion)
-*0.4 µL 10mM dNTPs
-*1 µL sense
-*1 µL antisense
-*0 µL DNA
-*0.2 µL phusion polymerase
-One for CFP and one for EYFP:
-*11.4µL milliQ water
-*4 µL buffer (5x Phusion)
-*0.4 µL 10mM dNTPs
-*1 µL sense (prefix primer)
-*1 µL antisense (antisense FP Fusion c-term tag)
-*2 µL DNA
-*0.2 µL Phusion polymerase
-One for CFP and one for GFP:
-*28.5µL milliQ water
-*10 µL buffer (5x Phusion)
-*1 µL 10mM dNTPs
-*2.5 µL sense (prefix primer)
-*2.5 µL antisense (antisense FP Fusion c-term tag)
-*2 µL DNA
-*0.5 µL Phusion polymerase
-Control: no polymerase (water= 29µL)
-'''Roxanne'''
-% agarose gel in TAE of the colony PCR
-E1= plate 1 of EYFP ligation
-E2= plate 2 of EYFP ligation
-(-)= no template
-(+)= EYFP/no pTet
-P1=plate 1 of pTet control ligation
-P2=plate 2 of pTet control ligation
-Lane
-#Ladder
-#E1-1
-#E1-2
-#E1-3
-#E1-4
-#E1-5
-#E2-1
-#E2-2
-#E2-3
-#E2-4
-#E2-5
-#+
-#–
-#P1-1
-#P1-2
-#P1-3
-#P1-4
-#P2-1
-#P2-2
-#P2-3
-[[Image:July_30-colonyPCR.png|250px]]
-Completed the maxipreps of pBAD, TetR, pLacI, mRBS,sRBS and Lumazine 2.  They were left at isopropanol precipitation.
-Restriction Digest
-dT-EcoRI & XbaI
-pTet-SAP
-pSB1A3-1 -EcoRI, Spe1
-n-terminal tag-XbaI, PstI
-Lumazine- XbaI, Spe1
-pSB1A3-2- XbaI, PstI
-C-terminal tag- EcoRI, Spe1
-{|
-|
-|'''dT'''
-|''pTet''
-|pSB1A3-1
-|'''N-term'''
-|''Lumazine''
-|pSB1A3-2
-|'''C-term'''
-|-
-|DNA
-|'''50 µL'''
-|''20 µL''
-|50 µL
-|'''20 µL'''
-|''20 µL''
-|20 µL
-|'''20 µL'''
-|-
-|10x buffer
-|'''7 µL'''
-|''3 µL''
-|7 µL
-|'''3 µL'''
-|''3 µL''
-|3 µL
-|'''3 µL'''
-|-
-|Enzyme 1
-|'''2 µL'''
-|''1 µL''
-|2 µL
-|'''1 µL'''
-|''1 µL''
-|1 µL
-|'''1 µL'''
-|-
-|Enzyme 2
-|'''2 µL'''
-|''0''
-|2 µL
-|'''1 µL'''
-|''1 µL''
-|1 µL
-|'''1 µL'''
-|-
-|milliQ water
-|'''9 µL'''
-|''6 µL''
-|9 µL
-|'''9 µL'''
-|''9 µL''
-|9 µL
-|'''9 µL'''
-|-
-|total
-|'''70 µL'''
-|''30 µL''
-|70 µL
-|'''30 µL'''
-|''30 µL''
-|30 µL
-|'''30 µL'''
-|}
-Prefix-N-terminal tag//gene
-			Gene//c-terminal-suffix
-Ran a 1x agarose gel of PCR products from earlier.
-Lane:
-#1kb ladder
-#Control
-#CFP-phusion
-#EYFP-phusion
-#CFP-econo taq
-#EYFP-econo taq
-[[Image:July_30_C-term_PCR.png|250px]]
-The PCR worked
-===July 31===
-'''Mackenzie + Lisza'''
-Gel extraction of c-term tag, n-term tag and lumazine.
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