Contents 1 August 1.1 August 3, 2010 1.2 August 3, 2010 Evening 1.3 August 4, 2010 1.4 August 6, 2010 1.5 August 6, 2010 Evening 1.6 Aug 9, 2010 1.7 Aug 9,2010 Evening 1.8 Aug 10, 2010 1.9 Aug 10, 2010 Evening 1.10 Aug 11, 2010 1.11 Aug 12, 2010 1.12 Aug 13, 2010 1.13 Aug 14, 2010 1.14 Aug 14, 2010 Evening 1.15 Aug 15, 2010 1.16 Aug 15, 2010 Evening 1.17 Aug 16, 2010 1.18 Aug 16, 2010 Evening 1.19 Aug 17, 2010 1.20 Aug 17, 2010 Evening 1.21 Aug 19, 2010 1.22 Aug 20, 2010 1.23 Aug 23, 2010

August

August 3, 2010

(in Lab: HB, AV, JV)

Objective: To restrict pBAD, sRBS, mRBS, TetR, dT and pTet for the assembly of pBAD-sRBS-TetR-dT-pTet

Method: Used Restriction of Plasmid DNA protocol.

• A front verctor was made made in sRBS ,mRBS, dT plasmids using EcoRI and Xbal enzymes
• pDNA that was cut out of plasmid for a front vector was pBAD, TetR, dT and pLacI using EcoRI and SpeI enzymes
• A back vector was made in sRBS and mRBS plasmids with PstI and SpeI
• pDNA that was cut out of plasmid for a back vector was TeTR and it was restricted with XbaI and PstI
  

Construct	pDNA	buffer	Enzymes
pBAD-sRBS/mRBS	pBAD	Red	EcoRI and SpeI
pBAD-sRBS/mRBS	sRBS	Orange	XbaI and EcoRI
pBAD-sRBS/mRBS	mRBS	Orange	XbaI and EcoRII
sRBS/mRBS-TetR	sRBS	Red	PstI and SpeI
sRBS/mRBS-TetR	mRBS	Red	PstI and SpeI
sRBS/mRBS-TetR	TetR	Tango	XbaI and PstI
TetR-dT	TetR	Red	EcoRI and SpeI
TetR-dT	dT	Orange	XbaI and EcoRI
dT-pTet	dT	Red	EcoRI and SpeI
dT-pTet	pTet	Orange	XbaI and EcoRI
pLAcI-sRBS/mRBS	pLacI	Red	EcoRI and SpeI
pLAcI-sRBS/mRBS	sRBS	Orange	XbaI and EcoRI
pLAcI-sRBS/mRBS	mRBS	Orange	XbaI and EcoRI
Mms6-PET28(a)	PET28(a)	Orange	NotI

• For all reactions
  • 158 (µL) Milli-Q H₂O
  • 10 (µL) Buffer
  • 0.5(µL) of each enzyme
  • 10 (µL) pDNA

Restriction was incubated for 1 hour at 37^oC


Objective: Run PCR of pBAD, TetR, dT, pLacI, and mms6.
Method: Used PCR thermocycler iGEM program 7

Component	Volume per tube (µL)	Master Mix (x6)
MilliQ H2O	41.8	250.8
10x Pfu Buffer with MgSO4	5	30
dNTPs	1	6
Forward Primer	0.5	3
Reverse Primer	0.5	3
Template DNA	1	-
Pfu DNA polymerase	0.2	-
Total	50	292.8

• Added 48.8 µL of Master Mix to each PCR reaction
  
  
  

Objective: Complete maxipreps of Lumazine (K249002), EYFP (E0030), and ECFP (E0020) and run on 1% agarose gel.
Method:

Lane	Sample	Components (µL)
1	1 kb ladder	2 loading dye (5x) + 0.5 ladder + 3.5 MilliQ H2O
2	ECFP	2 DNA + 2 loading dye (5x) + 2 MilliQ H2O
3	EYFP	2 DNA + 2 loading dye (5x) + 2 MilliQ H2O
4	Lumazine	2 DNA + 2 loading dye (5x) + 2 MilliQ H2O

• Ran at 100V for 42 minutes. Stained in EtBr for 15 minutes.
  

Results: AGAROSE GEL PICTURE


Objective: Ligate dT into pSB1C3.
Method:

• PCR amplify and purify both pSB1C3 and dT
• Restrict both with EcoRI and PstI
• Restrict pSB1C3 with DpnI
• Ligate pSB1C3 and dT
  

Restriction:

Restriction	MilliQ H2O (µL)	Buffer Orange (µL)	pDNA (µL)	Enzyme (µL)
pSB1C3	79.25	10	10	0.25 EcoRI + 0.25 PstI + 0.25 DpnI
pSB1C3 control	80	10	10	-
dT	79.50	10	10	0.25 EcoRI + 0.25 PstI
dT control	80	10	10	-

• Restriction were incubated at 37^oC for 90 minutes.
• Enzymes were heat killed for 20 minutes at 80^oC.

August 3, 2010 Evening

(in lab: KG, JS)

Objective: To run 1.5% agarose of restrictions: pBAD, sRBS, mRBS, TetR, dT and pTet for the assembly of pBAD-sRBS-TetR-dT-pTet

Method: Used a 1.5% agarose gel with 2 (µL) of loading dye and 10 (µL) of pDNA.

Lane	Contents	Result
1	1kb ladder
2	pTet (XbaI, EcoRI)	good
3	pTet (orange buffer)	---
4	dT (XbaI, EcoR)	no good
5	dT (orange buffer)	---
6	mRBS (SpeI, PstI)	good
7	mRBS (red buffer)	---
8	sRBS (SpeI, PstI)	good
9	sRBS (red buffer)	---
10	mRBS (XbaI, EcoRI)	good
11	mRBS (orange buffer)	---
12	sRBS (XbaI, EcoRl)	good
13	sRBS (orange buffer)	---
14	pet28(a)	good
15	100 bp ladder
16	PSB1C3	not good
17	PSB1C3 restriction digest	---

Lane	Contents	Result
1	TetR (EcorI, SpeI)	good
2	TetR (red buffer)	---
3	pLacI (EcoRI, SpeI)	good
4	pLacI (red buffer)	---
5	Mms6	can not tell
6	Mms6 control	---
7	TetR (Xbal, PstI)	good
8	TetR (tango buffer)	---
9	pBAD (EcoRI, SpeI)	good
10	pBAD (red buffer)	---
11	dT (EcoRI, SpeI)	good
12	dT (red buffer)	---
13	pLacI (2)	?
14	dT control	not good
15	dT restriction
16	100 bp ladder
17	dT PCR product	good
18	Mms6 PCR product	good
19	pBAD PCR product	good
20	pLacI PCR product	good

---

Objective: To ligate: pBAD-sRBS/mRBS, sRBS/mRBS-TetR, TetR-dT, dT-pTet, pLacI-sRBS/mRBS, Mms6-ptet28(a), dT-PSB1C3

Method: Ligation of Plasmid DNA

15 (µL) pDNA in plasmid, and 15 (µL) of pDNA biobrick)

August 4, 2010

(in lab: JV)

Objective: PCR analysis of ligation product of August 3, 2010

• Ligations
  • pBAD-mRBS
  • pBAD-SRBS
  • SRBS-tetR
  • mRBS-TetR
  • dt-pTet
  • mms6-pET-28a
  • dt-pSBIC3
  • pLacI-SRBS
• Controls
  • pBAD
  • TetR
  • TetR
  • pLacI
  • mms6

Method:
PCR: Thermocycler set to iGEM program 7

Component	1X(µL)	Master Mix(x16)(µL)
Milli-Q H2O	41.8	668.6
10x Pfu Buffer with MgSO4	5	80
dNTPs	1	16
Forward Primer	0.5	8
Reverse Primers	0.5	8
Template DNA	1	16
Pfu polymerase	0.2	3.2

2.5% agarose gel(1x TAE)

lane	contents	Successful Ligation ?
1	50bp ladder	---
2	dt pSBIC3	---
3	dt pTet	x
4	dt control	---
5	sRBS-TetR	x
6	mRBS-TetR	?
7	TetR control	---
8	pLacI-mRBS	x
9	pLacI-sRBS	?
10	pLacI control	---
11	Mms6 pET-28a	no band
12	Mms6 control	---
13	pBad-SRBS	x
14	pBad-mRBS	x
15	pBad control	---

• Ran at 100V for 70 minutes.

Results: AGAROSE GEL PICTURE


Objective: Transform the successful ligations

Method: used Competent Cell Transformation protocol

• changes:
  • used 50µL aliquottes of DH5&alpha
  • did not pipette up and down once, the cells were just swirled 3 times
  • added 400µL SOC media, shoock at 37⁰C for 90 min
  • platted 250µL and 150µL
    

Incubated from 12:00AM to 4;00 PM

results
contents	&250µL	150µL
dt-pTet	good	x
- control	x	x
mms6-pET-28a	good	good
dt-pSBIC3	x	x
mRBS-TetR	good	good
pLacI-mRBS	good	good
SRBS-TetR	x	x
pBAD-SRBS	good	good
+ contol	good	good

August 6, 2010

In Lab: JV

Objective: Put lumazine synthase gene and mms6 into pET-28(A) for future overexpression.

Method: Restrict mms6 from Mr. Gene with NotI. Large quantities of DNA can be used so a gel extraction of the part can be done. PCR lumazine synthase out of its backbone. Restrict off the extra DNA fragments from the PCR with NotI. Restrict pET-28A with notI. Ligate.

Restriction:

Restriction	MilliQ H2O (µL)	Buffer Orange (µL)	pDNA (µL)	Enzyme (µL)
mms6	799.5	100	100	1 NotI

PCR:

Component	1X(µL)
Milli-Q H2O	41.85
10x Pfu Buffer with MgSO4	5
dNTPs	1
Forward Primer (VF2)	0.5
Reverse Primers (VR)	0.5
Template DNA (Lumazine Synthase)	1
Pfu polymerase	0.2

2% Agarose Gel for Gel Extraction of mms6:

lane	sample	loaded
1	mms6 restricted with NotI	1 mL sample
2	mms6 unrestricted control	10(µL) sample, 2(µL)dye
3	50bp ladder	2(µL) ladder, 2(µL) dye, 8(µL) H20
3	1 kb ladder	2(µL) ladder, 2(µL) dye, 8(µL) H20

Gel ran for 60 minutes at 100 V. Small & faint band was slightly visible. Gel extraction was carried out using QIAGEN method. Eluted to 12 (µL).

August 6, 2010 Evening

Objective: Attempt colony pcr for rapid screening

Method: Followed two protocols from openwet

• Knight Protocol
  
  • place 20(µL) sterile H₂O in 0.6mL sterile tube
  • with P10 pipette set to 3(µL) dip tip into colony
  • place pipette tip into water and pipette up and down 20 times(this can be stored at 4⁰C for inoculation of overnight 5mL cultures)
    

• Endy Protocol
  • place 50(µL) sterile H₂O in 0.6mL sterile tube
  • with PLO pipette (set 3(µL)) dip sterile tip into colony
  • place pipette tip into water and pipette up and down 20 times

Knight cont'd	Endy cont'd
setup 20(µL) reaction	setup 20(µL) reaction
1(µL) colony suspension	2(µL) colony suspension
2(µL) 10x p.fu (+Mg SO4	2(µL) 10x p.fu (+Mg SO4
2(µL) dNTP	2(µL) dNTP
1.25(µL)VF2 Primer (10(µM)	0.75(µL)VF2 Primer (10(µM)
1.25(µL)VF Primer (10(µM)	0.75(µL)VF Primer (10(µM)
0.2(µL) Pfu polymerase	0.2(µL) Pfu polymerase
11.8 Milli-Q H2O	12.6 Milli-Q H2O

• as control for each rxn used equal volume of mRBS maxiprep

Knight cont'd	Endy cont'd
cycling conditions	cycling conditions
950C for 15 minutes	950C for 6 minutes
*940C for 30 seconds	**950C for 30 seconds
*560C for 30 seconds	**560C for 30 seconds
*680C for 1 minutes	**700C for 1 minutes
680C for 20 minutes	700C for 10 minutes

(*) were run 39 times
(**) were run 35 times

Made the following program (called COLONYY) Lid preheat 98⁰C

• 98⁰C for 15 minutes
• 98⁰C for 30 seconds
• 56⁰C for 30 seconds
• 68-70⁰C gradient for 1 minute
• 68-70⁰C gradient for 20 minute
• 4⁰C indefinte

bold selections were cycled 39 times

Objective: Analyzed PCR products on 2.5% TAE Agarose gel.

lane	sample	loaded
1	50bp ladder	1(µL) ladder, 1(µL) dye, 4(µL) H20
2	Knight control	5(µL) sample, 1(µL)dye
3	Knight colony	5(µL) sample, 1(µL)dye
4	Endy control	5(µL) sample, 1(µL)dye
5	Endy colony	5(µL) sample, 1(µL)dye
6	50bp ladder	1(µL) ladder, 1(µL) dye, 4(µL) H20
7	lumazine(justin's)	5(µL) sample, 1(µL)dye

Repeat gel with template controls

lane	sample	loaded
1	50bp ladder	0.5(µL) ladder, 2(µL) dye, 9.5(µL) H20
2	Endy Template	5(µL) colony suspension, 1(µL)dye, 4(µL) H20
3	Endy mRBS Control (PLR)	5(µL) sample, 1(µL)dye
4	Endy mRBS-TetR colony(PCR)	5(µL) sample, 1(µL)dye
4	mRBS template	0.5(µL) sample, 1(µL)dye, 4(µL) H20
5	Knight Template	0.25(µL) colony suspension, 1(µL)dye, 4(µL) H20
6	Knight mRBS control	5(µL) ladder, 1(µL) dye
7	Knight-mRBS-TetR colony	5(µL) sample, 1(µL)dye
1	1Kb ladder	0.5(µL) ladder, 2(µL) dye, 9.5(µL) H20

• ran at 100V for 75 minutes

Aug 9, 2010

(In Lab: HB)

Objective: Run PCR of mRBS-xylE I1 and I2 and lumazine.

Method:
PCR: Thermocycler set to iGEM program 7

Component	1X(µL)	Master Mix(x4)(µL)
Milli-Q H2O	41.8	167.2
10x Pfu Buffer with MgSO4	5	20
dNTPs	1	4
Forward Primer (VF2)	0.5	2
Reverse Primers (VR)	0.5	2
Template DNA	1
Pfu polymerase	0.2

Added 48.8µL Master Mix to each reaction tube.

Objective: Run 2% agarose gel to confirm PCR of mRBS-xylE I1 and I2 and lumazine worked.

lane	sample	loaded
1	50bp ladder	0.5(µL) ladder, 1(µL) dye, 6.5(µL) H20
2	mRBS-xylE I1	5(µL) sample, 2(µL)dye
3	mRBS-xylE I2	5(µL) sample, 2(µL)dye
4	Lumazine	5(µL) sample, 2(µL)dye

Ran at 100 V for 45 minutes. mRBS-xylE did not amplify while lumazine did amplify.

GEL PICTURE!

---

(In Lab: AV)

Objective: Prepared glycerol stocks & Miniprepped the following using the Qiagen Protocol.

pLacI-mRBS Colony 1

pLacI-mRBS Colony 2

pLacI-sRBS Colony 2

pLacI-sRBS Colony 3

pBAD-mRBS Colony 1

pBAD-mRBS Colony 2

pBAD-sRBS Colony 1

pBAD-sRBS Colony 2

dT-pTet Colony 1

dT-pTet Colony 3

mRBS-TetR Colony 1

mRBS-TetR Colony 3

---

Objective: To determine which of the previous ligations worked.

Method: Restricted with single cutter and double cutter.

Restriction Reaction (SINGLE)

Ingredient	Volume(µL)
MilliQ H20 Water	15.75
Orange Buffer (10x)	2
pDNA	2
EcoRI	0.25

Restriction Reaction (DOUBLE)

Ingredient	Volume(µL)
MilliQ H20 Water	15.50
Orange Buffer (10x)	2
pDNA	2
EcoRI	0.25
PstI	0.25

Unrestricted Control

Ingredient	Volume(µL)
MilliQ H20 Water	16
Orange Buffer (10x)	2
pDNA	2

DNA was restricted for 1 hour at 37^oC.

Analyzed results on a 2% agarose gel with 2 (µL) of loading dye and 10 (µL) of pDNA. Load order as follows:

Lane	Contents
1	1kb ladder
2	50 bp ladder
3	dT-pTet 1 DRD
4	dT-pTet 1 SRD
5	dT-pTet 1 URD
6	dT-pTet 3 DRD
7	dT-pTet 3 SRD
8	dT-pTet 3 URD
9	pLacI-mRBS 1 DRD
10	pLacI-mRBS 1 SRD
11	pLacI-mRBS 1 URD
12	pLacI-mRBS 2 DRD
13	pLacI-mRBS 2 SRD
14	pLacI-mRBS 2 URD
15	pLacI-sRBS 1 DRD
16	pLacI-sRBS 1 SRD
17	pLacI-sRBS 1 URD
18	pLacI-sRBS 3 DRD
19	pLacI-sRBS 3 SRD
20	pLacI-sRBS 3 URD

Lane	Contents
1	1 kb ladder
2	50 bp ladder
3	pBAD-mRBS 1 DRD
4	pBAD-mRBS 1 SRD
5	pBAD-mRBS 1 URD
6	pBAD-mRBS 2 DRD
7	pBAD-mRBS 2 SRD
8	pBAD-mRBS 2 URD
9	pBAD-sRBS 1 DRD
10	pBAD-sRBS 1 SRD
11	pBAD-sRBS 1 URD
12	pBAD-sRBS 2DRD
13	pBAD-sRBS 2 SRD
14	pBAD-sRBS 2 URD
15	mRBS-TetR 1 DRD
16	mRBS-TetR 1 SRD
17	mRBS-TetR 1 URD
18	mRBS-TetR 3 DRD
19	mRBS-TetR 3 SRD
20	mRBS-TetR 3 URD

GEL PICTURE!

---

Aug 9,2010 Evening

(In Lab: JV, AS)

Objective: To ligate: lumazine into vector upstream of dT. Lumazine and mms6 into pET28a.

Method:

• Restrictions
  
  • Restrict Lumazine wit EcoRI and SpeI (Red Buffer)
    
  • Restrict the dT with XbaI and EcoRI (Orange Buffer)
    
  • Restrict Lumazine Synthase with NotI (Red Buffer)
    
  Set up reactions as follows:
  

  Component	Volume (µL)
  MilliQ H2O	15.6 or 15.8
  Buffer	2
  pDNA	2
  Enzyme	0.20 + 0.20

  Incubated reactions for 60 minutes at 37^oC
  

• Ligation
  Reaction set up as follows:
  • T4 DNA ligase - 0.25µL
    
  • DNA 1 - 8µL
    
  • DNA 2 - 8µL
    
  • 10x Ligation Buffer - 2µL
    
  • MilliQ H₂O - 1.75µL
    
  Incubated reactions overnight at room temperature.

  Aug 10, 2010

  (In Lab: JV)
  

  Objective: Reran large gel from Aug 9/2010.
  

  Load order was as follows:

  Lane	Contents
  1	50 bp ladder
  2	pBAD-mRBS 2 URD
  3	pBAD-mRBS 2 SRD
  4	pBAD-mRBS 2 DRD
  5	pLacI-sRBS 3 URD
  6	pLacI-sRBS 3 SRD
  7	pLacI-sRBS 3 DRD
  8	pLacI-mRBS 2 URD
  9	pLacI-mRBS 2 SRD
  10	pLacI-mRBS 1 DRD
  11	dT-pTet 3 URD
  12	dT-pTet 3 SRD
  13	dT-pTet 3 DRD
  14	pBAD-mRBS 1 URD
  15	pBAD-mRBS 1 SRD
  16	pBAD-mRBS 1 DRD
  17	pLacI-sRBS 2 URD
  18	pLacI-sRBS 2 SRD
  19	pLacI-sRBS 2 DRD
  20	1 kb ladder

  
  

  Lane	Contents
  1	50 bp ladder
  2	pLacI-mRBS 1 URD
  3	pLacI-mRBS 1 SRD
  4	pLacI-mRBS 1 DRD
  5	dT-pTet 1 URD
  6	dT-pTet 1 SRD
  7	dT-pTet 1 DRD
  8	mRBS-TetR 3 URD
  9	mRBS-TetR 3 SRD
  10	mRBS-TetR 3 DRD
  11	mRBS-TetR 1 URD
  12	mRBS-TetR 1 SRD
  13	mRBS-TetR 1 DRD
  14	pBAD-sRBS 2 URD
  15	pBAD-sRBS 2 SRD
  16	pBAD-sRBS 2 DRD
  17	pBAD-sRBS 1 URD
  18	pBAD-sRBS 1 SRD
  19	pBAD-sRBS 1 DRD
  20	1 kb ladder

  
  

  GEL PICTURE!

  ---

  (In Lab: JV)
  

  Objective: Determine which ligations/transformations worked from 08/04/10.
  

  Method: Colony PCR.
  

  A: dT-pTet (1-10) B: pBAD-sRBS (1-10) C: pLacI-sRBS (1-10) D: pBAD-mRBS (1-10) E: mRBS-TetR (1-10) F: pLacI-mRBS (1-10)

  Pick colony with pipette set at 3(µL) Pipette colony up and dow in 20(µL) sterile Milli-Q water. Will use 96 well plate.

  PCR- Conditions: 1. 95^oC for 15 min 2. 98^oC for 20 sec 3. 55^oC for 40 sec 4. 72^oC for 2 min 5. 72^oC for 20 min 6. 4^oC infinitely

  Reaction Mixture -

  Method:
  PCR: Thermocycler set to iGEM program 7
  

  Component	1X(µL)	Master Mix(x65)(µL)
  Milli-Q H2O	10.9	708.5
  5X Phusion HF Buffer	4	260
  dNTPs	1	65
  Forward Primer (VF2)	1	65
  Reverse Primers (VR)	1	65
  Colony Template	2
  Phusion polymerase	0.17	11.05

  
  

  Controls: sRBS, pTET & mRBS - will PCR these to compare size using same conditions as above.

  GEL PICTURE!

  Aug 10, 2010 Evening

  (In Lab: ADS)
  

  Objective: PCR amplify xylE from mRBS-xylE for creation of xylE BioBrick
  

  Method: 20µL reactions
  

  PCR- Conditions: 1. Initial Denaturation 98^oC for 30 sec 2. Denaturation 98^oC for 10 sec 3. Anneal (51^oC, 55^oC,59.8^oC, 64.6^oC, 69.1^oC, 71^oC) for 30 sec 4. Extend 72^oC for 30 sec 5. Final Extend 72^oC for 10 min 6. Held 4^oC for 30 hours

  6 tubes in gradient PCR.

  Component	1X(µL)	Master Mix(x6.5)(µL)
  Milli-Q H2O	8.8	57.2
  5X Phusion HF Buffer	4	26
  dNTPs	2	13
  Forward Primer	2	13
  Reverse Primer	2	13
  Template DNA	1	6.5
  Polymerase	0.2	1.3

  
  

  Ran samples on 1.5% agarose gel (1X TAE) for 60 minutes at 100V.

  GEL PICTURE!

  Aug 11, 2010

  (In Lab: AS, JV, TF)
  

  Objective: Determine what transformations have the correct insert from Aug. 4, 2010.
  Method: Colony PCR. Changes - Used pipette tip instead of toothpick.
  PCR: Thermocycler set to iGEM PFUTEST
  

  1. pLacI-mRBS: 4 (A - E) 50(µL) 2. pBAD-mRBS: 5 (A - E) 20(µL) 3. mRBS-TetR: 6 (A -E) 20(µL) Controls - mRBS

  Component	1X(µL)	Master Mix(x7.5)(µL)
  Milli-Q H2O	9.8	73.5
  10x Pfu Buffer with MgSO4	2	15
  dNTPs	2	15
  Forward Primer (VF2)	2	15
  Reverse Primer (VR)	2	15
  Template DNA	2
  Pfu polymerase	0.2	1.5

  
  

  Added 18µL Master Mix to each reaction tube.

  ---

  (In Lab: ADS)
  

  Objective: Transform mRBS-xylE BioBrick into DH5alpha.
  

  Transformation -

  A) Thawed 50(µL) Sub-Cloning Efficiency DH5alpha Competent Cells on ice. B) Gently mixed cells and then aliquoted 100(µL) into chilled polypropylene tubes. C) Added 2(µL) of BioBrick to cells. Added 5(µL) of pUC19 DNA to 100(µL) cells to determine efficiency. D) Incubated cells on ice for 30 minutes. E) Heat shocked cells for 45 seconds in a 42^oC water bath. F) Placed on ice for 5 minutes. G) Added 0.4 mL of room temperature SOC medium. H) Shook at 225 rpm for 1 hour. I) Diluted control cells 1:100 with SOC medium. J) Spread 100(µL) of this dilution on LB-Amp agar plates K) Spread 50 and 250(µL) of experimental cells on LB-Cam agar plates. L) Incubated overnight at 37^oC

  Aug 12, 2010

  (In Lab: JV)
  

  Objective: Determine if Adam's colony PCRs' from Aug. 11, 2010 worked.
  Method: Samples were run on a 2.5% agarose gel (1X TAE) for 1 hour at 100V.
  

  GEL PICTURE!

  Results: Lanes 2, 3, 4 and 8 showed PCR amplification. Colonies chosen don't show the correct insert size.
  

  ---

  (In Lab: JV)
  Objective: Screen for colonies with the correct insert from Aug. 4, 2010 transformations.
  

  Method: Colony PCR. Changes - Used pipette tip instead of toothpick. Put colony in 20(µL) autoclaved Milli-Q water.
  PCR: Thermocycler set to iGEM PFUTEST
  

  1. pLacI-sRBS: A (11 - 17) 2. pBAD-sRBS: B (11 - 17) 3. dT-pTet: C (11 - 17) 4. pLacI-mRBS: D (11-17) 5. pBAD-mRBS: E (11-17) 6. mRBS-TetR: F (11-17)

  Controls - mRBS, pTet, sRBS

  Component	1X(µL)	Master Mix(x47)(µL)
  Milli-Q H2O	9.8	460.6
  10x Pfu Buffer with MgSO4	2	94
  dNTPs	2	94
  Forward Primer (VF2)	2	94
  Reverse Primer (VR)	2	94
  Template DNA (Cell Lysate)	2	94
  Pfu polymerase	0.2	9.4

  
  

  Added 18µL Master Mix to each reaction tube. Analyzed PCR products on 2.5% TAE gel. GEL PICTURE!!!

  ---

  (In Lab: ADS, KG)
  Objective: Perform PCR on lumazine, mms6, xylE plasmids with prefix and suffix primers (these will tell us exact size without subtracting VF2/VR regions). If right will ligate into pET28a plasmids.
  

  Method: Plasmids used included: 6 mms6 maxipreps. 4 lumazine maxipreps. 5 xylE maxipreps. 1 mRBS maxiprep
  PCR: Thermocycler set to iGEM Program #11 PFU - P/S
  

  PCR- Conditions: 1. Initial Denaturation 95^oC for 3 min 2. Denaturation 95^oC for 30 sec 3. Anneal (54^oC) for 30 sec 4. Extend 72^oC for 3 min 5. Final Extend 72^oC for 15 min 6. Held 4^oC infinitely (25 cycles)

  Component	1X(µL)	Master Mix(x16.5)(µL)
  Milli-Q H2O	9.8	161.7
  10x Pfu Buffer with MgSO4	2	33
  dNTPs	2	33
  Forward Primer (Prefix)	2	33
  Reverse Primer (Suffix Antisense)	2	33
  Template DNA (Cell Lysate)	2
  Pfu polymerase	0.2	3.3

  
  

  Added 18µL Master Mix to each reaction tube. Analyzed PCR products on 2.5% TAE gel run at 100 V for 35 minutes. GEL PICTURE!!!

  Aug 13, 2010

  (In Lab: AS)
  

  Objective: PCR amplify minipreps prepared on Aug 9/2010 to screen for properly assembled BioBricks.
  

  
  

  Component	1X(µL)	Master Mix(x12.5)(µL)
  Milli-Q H2O	41.85	523.1
  10x Pfu Buffer with MgSO4	5	62.5
  dNTPs	1	12.5
  Forward Primer (VF2)	0.5	6.25
  Reverse Primer (VR)	0.5	6.25
  Template DNA	1
  Pfu polymerase	0.15	1.888

  
  

  Added 49µL Master Mix to each reaction tube.

  ---

  (In Lab: AS)
  

  Objective: PCR amplify pSB1A3, pSB1T3 and pSB1C3 for use in future 3 part assembly and subsequent growth for glycerol stock.
  

  Component	1X(µL)	Master Mix(x13.5)(µL)
  Milli-Q H2O	14.9	52.15
  10x Pfu Buffer with MgSO4	2	7
  dNTPs	1	3.5
  Forward Primer (SB-prep-2)	0.7	2.45
  Reverse Primer (SB-prep-3p)	0.7	2.45
  Template DNA	0.5
  Pfu polymerase	0.2	0.7

  
  

  PCR- Conditions: 1. 94^oC for 30 sec 2. 94^oC for 30 sec 3. 55^oC for 30 sec 4. 68^oC for 3 min 5. 68^oC for 10 min 6. 4^oC infinitely (36 cycles)

  Added 19.5µL Master Mix to each reaction tube. Analyzed products on 1% TAE agarose gel which ran for 60 minutes at 100 V.

  GEL PICTURE!

  Aug 14, 2010

  (In Lab: AS)
  

  2.5% agarose gel(1x TAE)
  

  lane	contents
  1	pBAD-mRBS 1
  2	pBAD-mRBS 2
  3	pBAD-sRBS 1
  4	pBAD-sRBS 2
  5	mRBS-TetR 1
  6	mRBS-TetR 3
  7	50 bp Ladder
  8	dT-pTet 1
  9	dT-pTet 3
  10	pLacI-mRBS 1
  11	pLacI-mRBS 2
  12	pLacI-sRBS 2
  13	pLacI-sRBS 3
  14	MT
  15	MT
  16	MT
  17	MT
  18	MT
  19	MT
  20	MT
  21	No Lanes
  22	No Lanes
  23	No Lanes
  24	No Lanes
  25	No Lanes
  26	No Lanes
  27	No Lanes

  lane	contents
  1	K249001
  2	K249004
  3	K249005
  4	K249006
  5	MT
  6	K249008
  7	K249008 (Qiagen)
  8	K249014
  9	K249017
  10	50 bp Ladder
  11	1
  12	2
  13	3
  14	4
  15	5
  16	xylE-dT
  17	Lumazine-dT
  18	pLacI-sRBS
  19	MT
  20	MT
  21	MT
  22	MT
  23	MT
  24	MT
  25	MT
  26	MT
  27	MT

  GEL PICTURE!

  ---

  (In Lab: AS)
  

  Objective: Insert mms6 and lumazine into pET28a using NotI restriction site.
  

  Method: 1. Reserve 1(µL) of dirty PCR product for analysis. 2. Clean up PCR products using Qiagen prep. 3. Restrict 4 mms6 maxipreps and 4 lumazine maxipreps.
  

  Restriction Reactions:

  For lumazine and mms6 -

  Ingredient	1X(µL)	Master Mix(x8.5)(µL)
  MilliQ H20 Water	7.8	66.30
  Orange Buffer (10x)	2	17
  pDNA	10
  NotI	0.2	1.7

  Added 10 (µL) to each tube.

  For pET28a -

  Ingredient	Reaction Mix(µL)
  MilliQ H20 Water	4.8
  Orange Buffer (10x)	5
  pDNA	40
  NotI	0.2

  Incubated at 37^oC. Analyzed results on 2.5% TAE agarose gel which ran at 100 V for 50 minutes. GEL PICTURE!!!

  Results: Lost all the DNA in the column clean-up step and will have to re-do.
  

  Aug 14, 2010 Evening

  (In Lab: AS)
  

  Objective: Assemble mms6-dT and lumazine-dT using three antibiotic assembly.
  

  Method: 1. PCR amplify BioBricks (Prefix/Suffix) 2. Restrict BioBricks 3. Ligate BioBricks into psB1C3 4. Confirm ligation by PCR analysis (VF2/VR) 5. Transform ligation mixes 6. Screen colonies with Colony PCR
  

  PCR: Thermocycler set to iGEM program 11
  

  Component	1X(µL)	Master Mix(x10.5)(µL)
  Milli-Q H2O	10.8	113.4
  10x Pfu Buffer with MgSO4	2	21
  dNTPs	2	21
  Forward Primer (Prefix)	2	21
  Reverse Primers (Suffix Antisense)	2	21
  Template DNA	1
  Pfu polymerase	0.2	2.1

  
  

  Added 19 (µL) to each tube.

  Restriction Reactions:

  For lumazine and mms6 -

  Ingredient	1X(µL)	Master Mix(x5.5)(µL)
  MilliQ H20 Water	11.6	63.8
  Red Buffer (10x)	2	11
  pDNA	6
  EcoRI	0.2	1.1
  SpeI	0.2	1.1

  Added 14 (µL) to each tube.

  For dT -

  Ingredient	Reaction Mix(µL)
  MilliQ H20 Water	58
  Tango Buffer (10x)	10
  pDNA	30
  XbaI	1
  PstI	1

  For psB1C3 -

  Ingredient	Reaction Mix(µL)
  MilliQ H20 Water	70
  Orange Buffer (10x)	10
  pDNA	30
  EcoRI	1
  PstI	1
  DpnI	1

  Also cut lumazine, mms6 and dT with one enzyme for two part, PCR amplification and subsequent ligation into pSB1X3.

  For lumazine and mms6, CUT with SpeI -

  Ingredient	1X(µL)	Master Mix(x5.5)(µL)
  MilliQ H20 Water	15.8	86.9
  Tango Buffer (10x)	2	11
  pDNA	2
  SpeI	0.2	1.1

  Added 18 (µL) to each reaction.

  For dT, CUT with XbaI-

  Ingredient	Reaction Mix(µL)
  MilliQ H20 Water	79
  Tango Buffer (10x)	10
  pDNA	10
  XbaI	1

  Incubated at 37^oC for 1.5 hours. Heat killed enzymes for 20 minutes at 80^oC.

  Ligation Reactions:

  3 Part: Lumazine/mms6 + dT + psB1C3

  Ingredient	1X(µL)	Master Mix(x5.5)(µL)
  MilliQ H20 Water	11.0	64.9
  T4 Ligase Buffer (10x)	2	11
  Plasmid (psB1C3)	2	11
  Part 1 (Lumazine/mms6)	2
  Part 2 (dT)	2	11
  T4 DNA Ligase	0.2	1.1

  2 Part: Lumazine/mms6 + dT

  Ingredient	1X(µL)	Master Mix(x5.5)(µL)
  MilliQ H20 Water	13.8	75.9
  T4 Ligase Buffer (10x)	2	11
  Part 1 (Lumazine/mms6)	2
  Part 2 (dT)	2	11
  T4 DNA Ligase	0.2	1.1

  Added 18(µL) to each rxn tube. Incubated 1 hour and overnight at room temperature ( 25^oC).

  
  Screening via PCR amplification : Thermocycler set to iGEM program 11
  3 Part: Lum/mms6 + dT + pSB1C3

  Component	1X(µL)	Master Mix(x5.5)(µL)
  Milli-Q H2O	33.8	185.9
  10x Pfu Buffer with MgSO4	5	27.5
  dNTPs	2	11
  VF2 Primer	2	11
  VR Primer	2	11
  Template DNA	5
  Pfu polymerase	0.2	1.1

  
  

  Added 45 (µL) MM to each tube.

  2 Part: Lum/mms6 + dT

  Component	1X(µL)	Master Mix(x5.5)(µL)
  Milli-Q H2O	6.8	37.4
  10x Pfu Buffer with MgSO4	2	11
  dNTPs	2	11
  Prefix Primer	2	11
  Suffix Antisense Primer	2	11
  Template DNA	5
  Pfu polymerase	0.2	1.1

  
  

  Added 15 (µL) MM to each tube.

  Aug 15, 2010

  (In Lab: ADS)
  

  Objective: Insert mms6 and lumazine into pET28a using NotI restriction site.
  

  Method: 1. Reserve 1(µL) of dirty PCR product for analysis. 2. Clean up PCR products using Qiagen prep. 3. Restrict 3 mms6 maxipreps and 2 lumazine maxipreps.
  

  Restriction Reactions:

  For lumazine and mms6 -

  Ingredient	1X(µL)	Master Mix(x10.5)(µL)
  MilliQ H20 Water	11.8	123.9
  Orange Buffer (10x)	2	21
  pDNA	6
  NotI	0.2	2.1

  Added 14 (µL) to each tube.

  For pET28a -

  Ingredient	Reaction Mix(µL)
  MilliQ H20 Water	4.8
  Orange Buffer (10x)	5
  pDNA	40
  NotI	0.2

  Incubated at 37^oC for 1.5 hours. Heat killed enzymes at 80^oC for 20 minutes.

  Ligation Reactions:

  Ingredient	1X(µL)	Master Mix(x5.5)(µL)
  MilliQ H20 Water	13.9	75.9
  T4 Ligase Buffer (10x)	2	11
  Plasmid (pET28a)	2	11
  Cut out part (Lumazine/mms6)	2
  T4 DNA Ligase	0.2	1.1

  Added 18(µL) to each tube. Incubated 1 hour and overnight at room temperature.

  Aug 15, 2010 Evening

  (In Lab: AS)
  

  Objective: Assemble Lum-dT & mms6-dT using BioBrick standard assembly.
  

  Method: Obtain plasmid DNA from maxipreps.
  

  Restriction Reactions:

  For lumazine and mms6 -

  Ingredient	1X(µL)	Master Mix(x5.5)(µL)
  MilliQ H20 Water	7.6	41.8
  Red Buffer (10x)	2	11
  pDNA	10
  EcoRI	0.2	1.1
  SpeI	0.2	1.1

  For dT -

  Ingredient	Reaction Mix(µL)
  MilliQ H20 Water	38
  Orange Buffer (10x)	10
  pDNA	50
  XbaI	1
  EcoRI	1

  Incubated at 37^oC for 1.5 hours. Heat killed enzymes for 20 minutes at 80^oC.

  Ligation Reactions:

  Ingredient	1X(µL)	Master Mix(x5.5)(µL)
  MilliQ H20 Water	13.8	75.9
  T4 Ligase Buffer (10x)	2	11
  Plasmid (dT)	2	11
  Cut out part (Lumazine/mms6)	2
  T4 DNA Ligase	0.2	1.1

  Added 18(µL) MM to each rxn tube. Incubated at one hour and overnight at room temperature.

  Screening via PCR amplification : Thermocycler set to iGEM program 11
  

  Component	1X(µL)	Master Mix(x5.5)(µL)
  Milli-Q H2O	36.8	185.9
  10x Pfu Buffer with MgSO4	5	27.5
  dNTPs	2	11
  VF2 Primer	2	11
  VR Primer	2	11
  Template DNA	2
  Pfu polymerase	0.2	1.1

  
  

  Added 45 (µL) MM to each tube.

  Analyzed PCR products of BioBrick standard assembly; 3 part (or 3 antibiotic) assembly; and 3 part (3AB) Intermediate/2 part assembly on a 2% TAE agarose gel.

  GEL PICTURE!

  Aug 16, 2010

  (In Lab: KG)
  

  Objective: Confirm overnight ligations done on August 15, 2010.
  

  Method:
  

  Screening via PCR amplification : Thermocycler set to iGEM program 4
  

  Component	1X(µL)
  Milli-Q H2O	33.8
  10x Pfu Buffer with MgSO4	5
  dNTPs	2
  VF2 Primer	2
  VR Primer	2
  Template DNA	5
  Pfu polymerase	0.2

  
  

  3AB Master Mix

  Component	Master Mix(x5.5)(µL)
  Milli-Q H2O	185.9
  10x Pfu Buffer with MgSO4	27.5
  dNTPs	11
  Prefix Primer	11
  Suffix Primer	11
  Template DNA	2
  Pfu polymerase	1.1

  
  

  BBS/pSB1C3 Master Mix

  Component	Master Mix(x11)(µL)
  Milli-Q H2O	371.8
  10x Pfu Buffer with MgSO4	55
  dNTPs	22
  VF2 Primer	22
  VR Primer	22
  Template DNA
  Pfu polymerase	2.2

  
  

  Analyzed PCR products of overnight BioBrick standard assembly; 3 part (or 3 antibiotic) assembly; and 3 part (3AB) Intermediate/2 part assembly on a 2% TAE agarose gel.

  GEL PICTURE!

  Aug 16, 2010 Evening

  (In Lab: KG, AS)
  

  Objective: Restriction of PCR Products (mms6-dT, lumazine-dT). Restriction is necessary for ligation into plasmid backbone pSB1C3
  

  Method:
  

  Restriction Reactions:

  Ingredient	1X(µL)	Master Mix(x5.5)(µL)
  MilliQ H20 Water	7.6	41.8
  Orange Buffer (10x)	2	11
  pDNA	10
  PstI	0.2	1.1
  SpeI	0.2	1.1

  Incubated at 37^oC for 1.5 hours. Heat killed enzymes for 2 minutes at 80^oC.

  Ligation Reactions:

  Ingredient	1X(µL)	Master Mix(x5.5)(µL)
  MilliQ H20 Water	13.8	75.9
  T4 Ligase Buffer (10x)	2	11
  Plasmid Backbone (pSB1C3)	2	11
  pDNA	2
  T4 DNA Ligase	0.2	1.1

  ---

  (In Lab: KG)
  

  Objective: Transformations of insertions of mms6 or lumazine into pET28a.
  

  Method: Transformation of competent cells protocol.
  

  With the following changes: 1. 50(µL) aliquots of DH5alpha. 2. Added 400(µL) SOC media and put in shaker for 90 minutes at 37^oC. 3. Plated 150(µL) and 250(µL).

  Results: .
  

  Aug 17, 2010

  (In Lab: JV)
  

  Objective: Confirm ligations done on August 16, 2010.
  

  Method:
  Screening via PCR amplification : Thermocycler set to iGEM program 4
  

  Component	1X(µL)	Master Mix(x5.5)(µL)
  Milli-Q H2O	12.8	70.4
  10x Pfu Buffer with MgSO4	2	11
  dNTPs	1	5.5
  VF2 Primer	1	5.5
  VR Primer	1	5.5
  Template DNA	2
  Pfu polymerase	0.2	1.1

  
  

  Ran a 2% Agarose gel in 1X TAE buffer for 65 minutes at 100V.

  GEL PICTURE!

  Aug 17, 2010 Evening

  (In Lab: AS)
  

  Objective: Repeat ligation of mms6-dT and lumazine-dT to pSB1C3.
  Method: Use already restricted mms6-dT and lumazine-dT. Restrict pSB1C3 PCR product with EcoRI and PstI.
  

  Restriction Reactions:

  Ingredient	1X(µL)
  MilliQ H20 Water	28.6
  Orange Buffer (10x)	6
  pDNA (pSB1C3)	25
  PstI	0.2
  EcoRI	0.2

  Incubated at 37^oC for 1.5 hours. Heat killed enzymes for 20 minutes at 80^oC.

  Ligation Reactions:

  Ingredient	1X(µL)	Master Mix(x5.5)(µL)
  MilliQ H20 Water	13.8	75.9
  T4 Ligase Buffer (10x)	2	11
  Part 2 (pSB1C3)	2	11
  Part 1 (mms6-dT/Lum-dT)	2
  T4 DNA Ligase	0.2	1.1

  Added 18(µL) MM to each rxn tube. Incubated overnight at room temperature.

  ---

  Objective: Ligation confirmation by PCR. 2 different PCR reaction conditions were utilized. Believe PstI is not being heat inactivated.
  

  Method:
  PCR 1 - Show complete insertion of mms6-dT, lumazine-dT into pSB1C3. Both EcoRI and PstI ligations occurred.

  Component	1X(µL)	Master Mix(x11)(µL)
  Milli-Q H2O	33.8	371.8
  10x Pfu Buffer with MgSO4	5	55
  dNTPs	2	22
  Forward VF2 Primer	2	22
  Reverse VR Primer	2	22
  Template DNA	5
  Pfu polymerase	0.2	2.2

  
  

  Added 45(µL) MM to each rxn tube.

  PCR 2 - Show PstI is not heat killed and only EcoRI ligation occurred.

  Component	1X(µL)	Master Mix(x16)(µL)
  Milli-Q H2O	33.8	540.8
  10x Pfu Buffer with MgSO4	5	80
  dNTPs	2	32
  Forward VF2 Primer	2	32
  Reverse VR Primer	2	32
  Template DNA	5
  Pfu polymerase	0.2	3.2

  
  

  Added 45(µL) MM to each rxn tube.

  Aug 19, 2010

  (In Lab: JV)
  

  Objective: Determine if any transformations from Aug 16, 2010 have the correct insert.
  Method: Pick colonies and incubate at 37^oC in LB Media with Kan overnight. Use QIAGEN method to extract plasmid DNA. Restrict plasmid DNA to determine if mms6 or lumazine has correctly ligated into pET-28(A).
  

  Restriction Reactions:

  mms6 RESTRICTED -

  Ingredient	1X(µL)	Master Mix(x31)(µL)
  MilliQ H20 Water	15.75	488.25
  Red Buffer (10x)	2	62
  Template DNA
  Enzyme (EcoRV)	0.25	7.75

  mms6 UNRESTRICTED -

  Ingredient	1X(µL)	Master Mix(x31)(µL)
  MilliQ H20 Water	16	496
  Red Buffer (10x)	2	62
  Template DNA

  lumazine RESTRICTED -

  Ingredient	1X(µL)	Master Mix(x31)(µL)
  MilliQ H20 Water	15.75	488.25
  Tango Buffer (10x)	2	62
  Template DNA
  Enzyme (EcoRV)	.25	7.75

  lumazine UNRESTRICTED -

  Ingredient	1X(µL)	Master Mix(x31)(µL)
  MilliQ H20 Water	16	496
  Tango Buffer (10x)	2	62
  Template DNA

  Added 18(µL) to each restriction digest reaction. Incubated at 37^oC for 1.5 hours. Heat killed enzymes for 20 minutes at 80^oC.

  Samples were run on a 2% agarose gel in 1X TAE Buffer.

  GEL PICTURE!

  Aug 20, 2010

  (In Lab: JV)
  

  Objective: Determine if attempts to PCR amplify plasmid backbone were successful.
  

  Method: Ran samples on 1% agarose gel with 1X TAE buffer for 50 minutes at 100 V.
  

  Results: DNA concentration was good, however there was no evidence of an insert into pET-28(A).
  

  GEL PICTURE!!!

  Aug 23, 2010

  (In Lab: JV)
  

  Objective: Obtained part <partinfo>BBa_K118021</partinfo> and <partinfo>BBa_I716462</partinfo>.
  

  Method: Used competent cell transformation protocol.
  

  ---

  (In Lab: HB)
  

  Objective: Restrict 18 maxiprepped parts to quantify DNA.
  

  Method: Restrict all 18 parts and run on a 1% agarose gel with unrestricted parts.
  

  Restriction Reactions:

  Restriction Mix -

  Ingredient	1X(µL)	Master Mix(x19)(µL)
  MilliQ H20 Water	12.8	243.2
  Orange Buffer (10x)	2	38
  Template DNA	5
  EcoRI	0.2	3.8

  Unrestricted Mix -

  Ingredient	1X(µL)	Master Mix(x19)(µL)
  MilliQ H20 Water	13	247
  Orange Buffer (10x)	2	38
  Template DNA	5

  15(µL) added to each rxn tube. Incubated at 37^oC for 1.5 hours.

  GEL PICTURE!

  Retrieved from "http://2010.igem.org/Team:Lethbridge/Notebook/Lab_Work/August"