Team:Lethbridge/Notebook/Lab Work/July

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Here you can check out the work we have done in the lab, click on a month to take a look!

Contents

1 July 2010

1.1 July 3/2010

1.2 July 5/2010

1.3 July 5/2010 Evening

1.4 July 6/2010

1.5 July 8/2010

1.6 July 8/2010 - Evening

1.7 July 9/2010

1.8 July 10/2010

1.9 July 12/2010

1.10 July 12/2010 - Evening

1.11 July 13/2010

1.12 July 14/2010

1.13 July 15/2010

1.14 July 15/2010 Evening

1.15 July 16,2010

1.16 July 19, 2010

1.17 July 19, 2010 Evening

1.18 July 20, 2010

1.19 July 20, 2010 Evening

1.20 July 21, 2010

1.21 July 21, 2010 Evening

1.22 July 23, 2010

1.23 July 27, 2010

1.24 July 28, 2010

1.25 July 29, 2010

1.26 July 29, 2010 Evening

July 2010

July 3/2010

(In Lab: JS)

Objective: To restrict and ligate pBAD-TetR and YEPs into pSB1C3 backbone.

Method: Used Restriction of Plasmid DNA protocol and ligated the parts into pSB1C3.

July 5/2010

(In Lab: JV, AV, HB)

Objective: Run a 1% agarose gel of purified PCR samples from June 24/10

Method:

Lane Sample Components (µL)

1 1kb Ladder 0.5 Ladder + 2 Dye (6X) + 7.5 Milli-Q H₂O

2 1 - pBAD (A4) 1 DNA + 2 Dye (6X) + 7 Milli-Q H₂O

3 2 - pBAD (A5) 1 DNA + 2 Dye (6X) + 7 Milli-Q H₂O

4 3 - SRBS (A6) 1 DNA + 2 Dye (6X) + 7 Milli-Q H₂O

5 4 - SRBS (A7) 1 DNA + 2 Dye (6X) + 7 Milli-Q H₂O

6 5 - CFP Complete (A8) 1 DNA + 2 Dye (6X) + 7 Milli-Q H₂O

7 6 - SRBS (A10) 1 DNA + 2 Dye (6X) + 7 Milli-Q H₂O

8 7 - EYFP (B1) 1 DNA + 2 Dye (6X) + 7 Milli-Q H₂O

9 8 - N term tag (B2) 1 DNA + 2 Dye (6X) + 7 Milli-Q H₂O

10 9 - pSB NEYFP (B4) 1 DNA + 2 Dye (6X) + 7 Milli-Q H₂O

11 10 - pSB NEYFP (B5) 1 DNA + 2 Dye (6X) + 7 Milli-Q H₂O

12 11 - CFP (B6) 1 DNA + 2 Dye (6X) + 7 Milli-Q H₂O

13 12 - pBAD-TetR (B10) 1 DNA + 2 Dye (6X) + 7 Milli-Q H₂O

14 13 - D3 1 DNA + 2 Dye (6X) + 7 Milli-Q H₂O

15 14 - C term (D4) 1 DNA + 2 Dye (6X) + 7 Milli-Q H₂O

16 15 - C term (D5) 1 DNA + 2 Dye (6X) + 7 Milli-Q H₂O

17 16 - pLacI (D6) 1 DNA + 2 Dye (6X) + 7 Milli-Q H₂O

18 17 - NEYFP (E2) 1 DNA + 2 Dye (6X) + 7 Milli-Q H₂O

19 18 - CEYFP (E6) 1 DNA + 2 Dye (6X) + 7 Milli-Q H₂O

20 19 - CEYFP (E7) 1 DNA + 2 Dye (6X) + 7 Milli-Q H₂O

1 1kb ladder 0.5 Ladder + 2 Dye (6X) + 7.5 Milli-Q H₂O

2 20 - EYFP (E8) 1 DNA + 2 Dye (6X) + 7 Milli-Q H₂O

3 21 - EYFP (E9) 1 DNA + 2 Dye (6X) + 7 Milli-Q H₂O

4 22 - EYFP (E10) 1 DNA + 2 Dye (6X) + 7 Milli-Q H₂O

5 23 - ECFP (F1) 1 DNA + 2 Dye (6X) + 7 Milli-Q H₂O

6 24 - ECFP (F2) 1 DNA + 2 Dye (6X) + 7 Milli-Q H₂O

7 25 - ECFP (F3) 1 DNA + 2 Dye (6X) + 7 Milli-Q H₂O

8 26 - pBAD-TetR (F4) 1 DNA + 2 Dye (6X) + 7 Milli-Q H₂O

9 27 - pBAD-TetR (F5) 1 DNA + 2 Dye (6X) + 7 Milli-Q H₂O

10 28 - EYFP (G1) 1 DNA + 2 Dye (6X) + 7 Milli-Q H₂O

11 29 - pSB CEYFP (G4) 1 DNA + 2 Dye (6X) + 7 Milli-Q H₂O

12 30 - pBAD (1) (G6) 1 DNA + 2 Dye (6X) + 7 Milli-Q H₂O

13 31 - pBAD (2) (G7) 1 DNA + 2 Dye (6X) + 7 Milli-Q H₂O

14 32 - N term tag (G8) 1 DNA + 2 Dye (6X) + 7 Milli-Q H₂O

15 33 - lumazine (G9) 1 DNA + 2 Dye (6X) + 7 Milli-Q H₂O

Ran gel at 100V for 45 minutes.

Results:

July 5/2010 Evening

Objective: To over-express CFP complete in DH5α

Method:

1) Inoculated 6mL culture with ampicillin and glycerol stock A6-May 13,2010 CFP complete 2) Went in the shaker at 6:50pm

July 6/2010

(In lab: JV, AV, HB)
Objective: To continue the over-expression of CFP complete in DH5α

Method:
1) Put both cultures (taken out of shaker at 9:15am) and put them in 500mL of LB w/ Amp.
2) initial OD was 0.071 (600λ)

Issue:

After checking the the sequencing it was evident that our promotor is always off. It is turned off by the product of the gene TetR. Which is not part of our construct.

Time (hours) OD (600λ)

0 0.071

1 0.390

1.5(T₀) 0.606

2.5 (T₁) 1.250

3.5(T₂) 3.04

4.5(T₃) 2.75

Results: Ran samples on a 15% SDS-page. The gel did not show any signs of over-expression.

Objective: To determine if maxipreps were successful via restriction by NotI and running on 1% Agarose gel

Method:
1) Restriction:

Component Volume (µL)

DNA 0.071

Buffer Orange 0.390

NotI 0.606

MilliQ H₂O 1.250

Incubated for 1 hour at 37^oC. Heat killed on heat block at 80^oC for 20 mins.

2) 1% Agarose gel

Lane Sample Volume (µL)

1 1 kb Ladder 0.5 Ladder + 2 Loading dye (6x) + 7.5 MilliQ H₂O

2 PET28a restricted 8 DNA + 2 Loading dye (6x)

3 PET28a 8 DNA + 2 Loading dye (6x)

4 Lumazine restricted 8 DNA + 2 Loading dye (6x)

5 Lumazine 8 DNA + 2 Loading dye (6x)

6 mms6 restricted 8 DNA + 2 Loading dye (6x)

7 mms6 8 DNA + 2 Loading dye (6x)

8 xylE restricted 8 DNA + 2 Loading dye (6x)

9 xylE 8 DNA + 2 Loading dye (6x)

10 Empty Empty

Ran at 100V for 80 mins.

Results:

More to fill in, but Anthony does not understand the stuff written in the lab book

July 8/2010

(In lab: JV, AV, HB, HS)
Objective:

Anthony does not understand the stuff written in the lab book.

July 8/2010 - Evening

(In lab: KG)
Objective:To transform TetR in pSB1A2 plasmid (BBa_C0040 - 2010 iGEM Distribution Kit Plate Well 4A) and pTetR in pSB1A2 plasmid (BBa_R0040 - 2010 iGEM Distribution Kit Plate Well 6) into DH5α.

Method:Used the Competent Cell Transformation Protocol as well as transformed pUC19 as a positive control.

Results:

Plate Number of Colonies

50 µL TetR 0

200 µL TetR 5

50 µL pTetR 4

200 µL pTetR 34

50 µL pUC19 3

200 µL pUC19 4

July 9/2010

(In lab: JV)
Objective:To overexpress pLacI-mRBS-mms6-dT construct.

Method:Used the Overexpression Protocol.

Time (hours) OD (600λ)

0 0.052

1 0.111

2 0.315

2.5 0.449

3(T₀) 0.772

4(T₁) 2.22

5(T₂) 2.06

6(T₃) 2.50

Samples were run on a 15% SDS PAGE for 20 minutes at 80V and 1 hour at 200V.

SDS PAGE picture!!!!!!!!!!!

July 10/2010

(In lab: JV)
Objective:To determine if maxipreps finished on July 9th and 10th have significant concentrations of DNA.

Method:Run 1% Agarose gel

Lane Sample Components (µL)

1 dT 2 DNA + 2 loading dye (6x) + 6 MilliQ H₂O

2 mms6 (1) 2 DNA + 2 loading dye (6x) + 6 MilliQ H₂O

3 mms6 (2) 2 DNA + 2 loading dye (6x) + 6 MilliQ H₂O

4 pBAD-TetR (1) 2 DNA + 2 loading dye (6x) + 6 MilliQ H₂O

5 pBAD-TetR (2) 2 DNA + 2 loading dye (6x) + 6 MilliQ H₂O

6 mRBS 2 DNA + 2 loading dye (6x) + 6 MilliQ H₂O

7 pLacI 2 DNA + 2 loading dye (6x) + 6 MilliQ H₂O

8 sRBS 2 DNA + 2 loading dye (6x) + 6 MilliQ H₂O

9 1 kb Ladder 0.5 ladder + 2 loading dye (6x) + 7.5 MilliQ H₂O

10 Empty Empty

Ran at 100V for 40 minutes. Strained in EtBr for 10 minutes.

Results:

July 12/2010

(In lab: AV,HB,JV)
Objective: Maxiprep pLacI (A2) from glycerol stock.

Method: Used Maxiprep Protocol. Cell pellet weighed 1.02g.

Objective: Add dT to the end of mms6, xylE, and lumazine.
Method:
1) Restrict "Part 1" BioBricks: mms6, xylE, and lumazine with EcoRI and SpeI.

Component Volume (µL)

pDNA 2

Red Buffer 2

EcoRI 0.25

SpeI 0.25

MilliQ H₂O 15.5

2) Restrict "Part 2" BioBrick: dT with EcoRI and XbaI.

Component Volume (µL) 2.5x Volume (µL)

pDNA 2 5

Orange Buffer 2 5

EcoRI 0.25 0.625

XbaI 0.25 0.625

MilliQ H₂O 15.5 38.75

3) 1% Agarose gel (1x TAE) of restricted DNA

Lane Sample Component (µL)

1 1 kb Ladder 0.5 ladder + 2 loading dye (6x) + 7.5 MilliQ H₂O

2 dT 8 DNA + 2 loading dye (6x)

3 dT restricted 8 DNA + 2 loading dye (6x)

4 mms6 8 DNA + 2 loading dye (6x)

5 mms6 restricted 8 DNA + 2 loading dye (6x)

6 xylE 8 DNA + 2 loading dye (6x)

7 xylE restricted 8 DNA + 2 loading dye (6x)

8 Lumazine 8 DNA + 2 loading dye (6x)

9 Lumazine restricted 8 DNA + 2 loading dye (6x)

10 Empty Empty

Ran at 100V for 43 minutes. Stained in EtBr for 10 minutes.
Results:

4) Ligate restricted dT to the ends of the "Part 1" Biobricks.

July 12/2010 - Evening

(In lab: KG,TF)
Objective: Continue with addition of dT to the ends of mms6, xylE, and lumazine.

Method:

4) Ligate restricted dT to the ends of the "Part 1" Biobricks.
dT to mms6:

Component Volume (µL)

T4 DNA Ligase 0.25

10x T4 Ligation Buffer 2

dT 8.3

mms6 9.2

MilliQ H₂O 0.25

dT to xylE:

Component Volume (µL)

T4 DNA Ligase 0.25

10x T4 Ligation Buffer 2

dT 8.3

xylE 3.4

MilliQ H₂O 6.05

dT to lumazine:

Component Volume (µL)

T4 DNA Ligase 0.25

10x T4 Ligation Buffer 2

dT 8.3

lumazine 3.35

MilliQ H₂O 6.1

Ligations were incubated at room temperature overnight.

Objective: Prepare mastermix for four PCR reactions for following day.

Method:

Component Volume (µL) 5x Volume (µL)

10mM dNTPs 1 5

5x Phusion Buffer 4 20

Forward Primer (VF2) 1 5

Reverse Primer (VR) 1 5

MilliQ H₂O 10.8 54

July 13/2010

(in lab: JV)
Objective: To determine if ligations of previous day (July 12/2010) were successful.
Method:
1) Use prepared mastermix to run a PCR of ligated mms6-dT, ligated xylE-dT, ligated lumazine-dT, and control-dT
- To each PCR tube, add 17.8&mirco;L of mastermix, 0.2µL of Phusion DNA polymerase, and 2µL of template DNA.
- Ran PCR's for 36 cycles using the iGEM preset.

2) 2% Agarose gel

Lane Sample Components (µL)

1 50 bp Ladder 1 ladder + 2 loading dye (6x) + 7 MilliQ H₂O

2 mms6-dT 8 PCR product + 2 loading dye (6x)

3 xylE-dT 8 PCR product + 2 loading dye (6x)

4 lumazine-dT 8 PCR product + 2 loading dye (6x)

5 dT 8 PCR product + 2 loading dye (6x)

6 Empty Empty

7 Empty Empty

8 Empty Empty

9 Empty Empty

10 Empty Empty

Ran at 100V for __ minutes. Stained in EtBr for 10 minutes.

Results:

July 14/2010

(in lab:J.S, K.G )
Objective: Inoculate culture for maxi prep with placI
Method: To a 450mL solution of LB media 4.5(µL) of ampicillin was added with glycerol placI aseptically.

(in lab:J.S, K.G )
Objective: Restriction of dT and mms6
protocol
1)

Component Volume (µL)

Buffer&* 2

10x T4 Milli Q H₂O 15.5

pDNA** 2

Restriction enzymes*** 0.25

* Orange buffer was used for dT, and Red buffer was used for mms6
**pDNA was dT and mms6
***Restriction enzymes for dT were XbalI and EcoRI, and for mms6 SpeI and EcoRI
2) Reaction was incubated at 37⁰C for 1 hour. Start 8:50pm till 9:50pm After incubation reaction was heat shocked at 80⁰C for 20 minutes

(in lab:J.S, K.G )
Objective: Ligation of dT to each of mms6, xylE and lumazine.
protocol
1)
dT to mms6:

Component Volume (µL)

T4 DNA Ligase 0.25

10x T4 Ligation Buffer 2

dT 8.3

mms6 9.2

MilliQ H₂O 0.25

dT to xylE:

Component Volume (µL)

T4 DNA Ligase 0.25

10x T4 Ligation Buffer 2

dT 8.3

xylE 3.4

MilliQ H₂O 6.05

dT to lumazine:

Component Volume (µL)

T4 DNA Ligase 0.25

10x T4 Ligation Buffer 2

dT 8.3

lumazine 3.35

MilliQ H₂O 6.1

2) Incubated reaction overnight at room temperature

July 15/2010

(in lab: AV)
Objective: Maxiprep pLacI and mms6.

component pallet weight (g)

mms6 1.02

pLacI 1.54

July 15/2010 Evening

(in lab: AV)
Objective: transform ligations from July 14 and July 12,2010;xylE/dt, mms6/dt lumazine/dt into DH5&alpha. Also to transform mms6 from July 6 and July 10 into Bl21(DE3)
Protocol: to transform competent cells see protocol:[1]
* cells were incubated on ice for 30 minutes started at 7:00-7:30. **incubated for 1 hour from 8:00 till 9:00

Results

plate # of colonies Components (µL)

1 200µL xylE-dt July 12 0

2 200µL mms6-dt July 12 1

3 200µL lumazine-dt 1

4 200µL mms6 maxiprep July 6 Lawn

5 200µL positve control puC19 into BL21(DE3) Lawn

6 200µL xylE-dt July 14 0

7 200µL mms6-dt July 14 120

8 200µL lumazine-dt July14 0

9 200µL mms6 maxiprep July 10 .

*because of a shortage of plates not all transformations were plated at 50µL and 200µL

July 16,2010

(in lab: M.C, D.M)
Objective: PCR amplify mms6-dT, xylE-dT, lumazine-dT legations along with dT maxi prep for comparison
Protocol:
Master Mix

Component Volume (µL)

Milli-Q H₂O< 54

Pfu Buffer + MgSO₄ 20

10 mM dMTPs 5

forward primer 5

reverse primer 5

89 µL TOTAL--> 17.8 into each PCR reaction
+ 2 µL ligation + 2 µL Pfu polymerase
Ran iGem-ligTest in thermocycler

July 19, 2010

(in lab: A.V, H.B)
Objective: Purification pf pLacI and mms6 maxipreps done on July 14 and 16, 2010 using the biobasic protocol for purification of PCR products.
Objective: Add pLacI to sRBS and add dT to xylE and lumazine
Method:
Restrict pLacI, xylE and lumazine with SpeI and EcoRI and restrict dT and sRBS with EcorI and XbaI
1)Restrictions:
pLacI, xylE, and Lumazine

Component Volume (µL)

Milli-Q H₂O 15.5

Red Buffer₄ 2

SpeI 0.25

EcoRI 0.25

pDNA 2

dT

Component Volume (µL)

Milli-Q H₂O 38.75

Orange Buffer₄ 5

XbaI 0.625

EcoRI 0.625

pDNA 5

sRBS

Component Volume (µL)

Milli-Q H₂O 15.5

Red Buffer₄ 2

xBal 0.25

EcoRI 0.25

pDNA 2

Restriction incubation at 37.5⁰C started at 11:55am. Ended at 12:55pm Heat killed enzymes at 80⁰C for 20 minutes
2)Restrictions were run on a 1% agarose gel (1 X TAE) 1% Agarose gel

Lane Sample Components (µL)

1 1kB Ladder 0.5 ladder + 2 loading dye (5x) + 5.5 MilliQ H₂O

2 pLacI 6 pDNA + 2 loading dye (5x)

3 pLacI restriction digest 6 pDNA + 2 loading dye (5x)

4 sRBS 6 pDNA + 2 loading dye (5x)

5 sRBS restriction digest 6 pDNA + 2 loading dye (5x)

6 xylE 6 pDNA + 2 loading dye (5x)

7 xylE restriction digest 6 pDNA + 2 loading dye (5x)

8 lumazine 6 pDNA + 2 loading dye (5x)

9 lumazine restriction digest 6 pDNA + 2 loading dye (5x)

10 dT 6 pDNA + 2 loading dye (5x)

11 dT restriction digest 6 pDNA + 2 loading dye (5x)

Gel ran for 70 minutes at 100V and was stained in EtBr for 10 minutes
Results:

Results after quantifying restriction digest

Lane Content Quantity of DNA (ng/µL)

1 1kB Ladder 12.5

3 pLacI restriction digest 5.21

5 sRBS restriction digest 3.72

7 xylErestriction digest 3.36

9 Lumazine restriction digest 2.63

11 dT restriction digest 2.98

July 19, 2010 Evening

(K.G)
Objective: Ligate together rbs-xylE and dT, lumazine and dT, also pLacI and sRBS.

Method: All ligation mixes had:

10X T4 Ligation Buffer
Milli-Q H₂O to fill to 20µL
T4 DNA Ligase
20ng plasmid DNA

Ligations were left over-night at room temperature.

(J.V.)
Objective:Determine the results of the transformations done by K.G. on July 15/2010.

Method: Inoculate 5mL LB media w/Amp and colony. Incubated over night at 37^oC.

Results: Lumazine Synthase with dT ligated onto it grew.

July 20, 2010

(AV, HB)
Objective:Miniprep lumazine-dt and 4 N-terminus tags and analyze.

Method:

Use boiling lysis miniprep to isolate plasmid DNA.
PCR amplify BioBrick part to determine if the correct DNA was isolated.
Ran a 1% Agarose gel to visualize PCR products.

Results: Agarose gel did not show any bands.

July 20, 2010 Evening

(AV, HB)
Objective:Transform ligation done on July 19, 2010 in to competent DH5α cells.

xylE-dT
lumazine synthase-dT
pLacI-sRBS
EYFP and ECFP

Method:

Use Competent Cell Transformation

Results:

xylE-dT no colonies
pLacI-sRBS
EYFP
lumazine-dT
ECFP

July 21, 2010

(JV)
Objective: Isolate plasmid DNA from pTet, TetR, pET-28(a).
Method:

Used Maxiprep protocol.

(JV)

Objective: To induce over-expression of pLacI-RBS-Mms6-dt in BL21(DE3) cells.

Method: Used Overexpression.
Ran SDS gel for 78 minutes at 200V

July 21, 2010 Evening

(TF, AS)
Objective: Colony PCR to test for insertions of BioBrick construction, and for quality control of parts received from the Registry. Also to prepare DNA to be sent away for sequencing.
Method:
-Ran PCR's
-Ran 2% Agarose gel for:.

K249001

K249004

K249005

K249006

K249008

K249014

K249017

mms6-dt

lumazine-dt

ECFP

EYFP

N-term tag

xylE-dt

SRBS-pLacI

dt

pTet maxiprep

pET-28(a) maxiprep

TetR maxiprep

Gel ran at 100V for 60minutes.
Results: The PCR's did not work. However, the maxipreps show DNA

July 23, 2010

(JV)
Objective: Insert xylE, Mms6, and lumazine synthase into pET-28(a).
Method: A restriction digest was performed on xylE and Mms6 and ran for 90 minutes at 37 C
Results:Did not see any cut out biobricks.

July 27, 2010

(in lab: JV, AV, HB)

Objective: To miniprep the overnight cultures of the parts ordered from the Parts Registry and to test the efficiency of the Qiagen Miniprep Kit.

Method:
The following parts were miniprep'd using Boiling Lysis Plasmid Preparation:

dT (control)
E0020 (ECFP)
E0030 (EYFP)
K249001
K249004
K249005
K249006
K249008
K249014
K249017

The following parts were miniprep'd using Qiagen Miniprep Kit:

K249005
K249008

Results: Qiagen Miniprep Kit produced a comparable quantity of DNA to Boiling Lysis Plasmid Preparation. Since the Qiagen Miniprep Kit is faster and easier, all minipreps will be done using the Qiagen Miniprep Kit.

July 28, 2010

(in lab: JV)

Objective: To create a large quantity of pSB1C3 plasmid.

Method: PCR amplified pSB1C3 received from iGEM headquarters.

July 29, 2010

(in lab: HB, AV)

Objective: To PCR amplify the minipreps on July 27, 2010.

Method: PCR amplified the 11 minipreps and dT control.

Results: PCR amplification was successful and 0.2 µL of polymerase will be used per PCR reaction in the future.

Objective: To maxiprep EYFP (E0030), ECFP (E0020), and Lumazine Synthase (K249002).

Method: Used Maxiprep

Cell Pellet Weight (g)

ECFP 2.32

EYFP 2.00

Lumazine Synthase 2.66

July 29, 2010 Evening

(in lab: KG)

Objective: Run PCR products from the morning on a 2% agarose gel.

Method: Ran 2% agarose gel of the 11 minipreps and dT control and stained in EtBr for 17 minutes.
Results:PCR amplification of last year's parts not consistent with sizes listed on registry.

Lane	Sample	Components (µL)
1	1kb Ladder	0.5 Ladder + 2 Dye (6X) + 7.5 Milli-Q H₂O
2	1 - pBAD (A4)	1 DNA + 2 Dye (6X) + 7 Milli-Q H₂O
3	2 - pBAD (A5)	1 DNA + 2 Dye (6X) + 7 Milli-Q H₂O
4	3 - SRBS (A6)	1 DNA + 2 Dye (6X) + 7 Milli-Q H₂O
5	4 - SRBS (A7)	1 DNA + 2 Dye (6X) + 7 Milli-Q H₂O
6	5 - CFP Complete (A8)	1 DNA + 2 Dye (6X) + 7 Milli-Q H₂O
7	6 - SRBS (A10)	1 DNA + 2 Dye (6X) + 7 Milli-Q H₂O
8	7 - EYFP (B1)	1 DNA + 2 Dye (6X) + 7 Milli-Q H₂O
9	8 - N term tag (B2)	1 DNA + 2 Dye (6X) + 7 Milli-Q H₂O
10	9 - pSB NEYFP (B4)	1 DNA + 2 Dye (6X) + 7 Milli-Q H₂O
11	10 - pSB NEYFP (B5)	1 DNA + 2 Dye (6X) + 7 Milli-Q H₂O
12	11 - CFP (B6)	1 DNA + 2 Dye (6X) + 7 Milli-Q H₂O
13	12 - pBAD-TetR (B10)	1 DNA + 2 Dye (6X) + 7 Milli-Q H₂O
14	13 - D3	1 DNA + 2 Dye (6X) + 7 Milli-Q H₂O
15	14 - C term (D4)	1 DNA + 2 Dye (6X) + 7 Milli-Q H₂O
16	15 - C term (D5)	1 DNA + 2 Dye (6X) + 7 Milli-Q H₂O
17	16 - pLacI (D6)	1 DNA + 2 Dye (6X) + 7 Milli-Q H₂O
18	17 - NEYFP (E2)	1 DNA + 2 Dye (6X) + 7 Milli-Q H₂O
19	18 - CEYFP (E6)	1 DNA + 2 Dye (6X) + 7 Milli-Q H₂O
20	19 - CEYFP (E7)	1 DNA + 2 Dye (6X) + 7 Milli-Q H₂O
1	1kb ladder	0.5 Ladder + 2 Dye (6X) + 7.5 Milli-Q H₂O
2	20 - EYFP (E8)	1 DNA + 2 Dye (6X) + 7 Milli-Q H₂O
3	21 - EYFP (E9)	1 DNA + 2 Dye (6X) + 7 Milli-Q H₂O
4	22 - EYFP (E10)	1 DNA + 2 Dye (6X) + 7 Milli-Q H₂O
5	23 - ECFP (F1)	1 DNA + 2 Dye (6X) + 7 Milli-Q H₂O
6	24 - ECFP (F2)	1 DNA + 2 Dye (6X) + 7 Milli-Q H₂O
7	25 - ECFP (F3)	1 DNA + 2 Dye (6X) + 7 Milli-Q H₂O
8	26 - pBAD-TetR (F4)	1 DNA + 2 Dye (6X) + 7 Milli-Q H₂O
9	27 - pBAD-TetR (F5)	1 DNA + 2 Dye (6X) + 7 Milli-Q H₂O
10	28 - EYFP (G1)	1 DNA + 2 Dye (6X) + 7 Milli-Q H₂O
11	29 - pSB CEYFP (G4)	1 DNA + 2 Dye (6X) + 7 Milli-Q H₂O
12	30 - pBAD (1) (G6)	1 DNA + 2 Dye (6X) + 7 Milli-Q H₂O
13	31 - pBAD (2) (G7)	1 DNA + 2 Dye (6X) + 7 Milli-Q H₂O
14	32 - N term tag (G8)	1 DNA + 2 Dye (6X) + 7 Milli-Q H₂O
15	33 - lumazine (G9)	1 DNA + 2 Dye (6X) + 7 Milli-Q H₂O

Time (hours)	OD (600λ)
0	0.071
1	0.390
1.5(T₀)	0.606
2.5 (T₁)	1.250
3.5(T₂)	3.04
4.5(T₃)	2.75

Component	Volume (µL)
DNA	0.071
Buffer Orange	0.390
NotI	0.606
MilliQ H₂O	1.250

Plate	Number of Colonies
50 µL TetR	0
200 µL TetR	5
50 µL pTetR	4
200 µL pTetR	34
50 µL pUC19	3
200 µL pUC19	4

Time (hours)	OD (600λ)
0	0.052
1	0.111
2	0.315
2.5	0.449
3(T₀)	0.772
4(T₁)	2.22
5(T₂)	2.06
6(T₃)	2.50

Component	Volume (µL)
pDNA	2
Red Buffer	2
EcoRI	0.25
SpeI	0.25
MilliQ H₂O	15.5

Component	Volume (µL)	2.5x Volume (µL)
pDNA	2	5
Orange Buffer	2	5
EcoRI	0.25	0.625
XbaI	0.25	0.625
MilliQ H₂O	15.5	38.75

Lane	Sample	Component (µL)
1	1 kb Ladder	0.5 ladder + 2 loading dye (6x) + 7.5 MilliQ H₂O
2	dT	8 DNA + 2 loading dye (6x)
3	dT restricted	8 DNA + 2 loading dye (6x)
4	mms6	8 DNA + 2 loading dye (6x)
5	mms6 restricted	8 DNA + 2 loading dye (6x)
6	xylE	8 DNA + 2 loading dye (6x)
7	xylE restricted	8 DNA + 2 loading dye (6x)
8	Lumazine	8 DNA + 2 loading dye (6x)
9	Lumazine restricted	8 DNA + 2 loading dye (6x)
10	Empty	Empty

Component	Volume (µL)
T4 DNA Ligase	0.25
10x T4 Ligation Buffer	2
dT	8.3
mms6	9.2
MilliQ H₂O	0.25

Component	Volume (µL)	5x Volume (µL)
10mM dNTPs	1	5
5x Phusion Buffer	4	20
Forward Primer (VF2)	1	5
Reverse Primer (VR)	1	5
MilliQ H₂O	10.8	54

Component	Volume (µL)
Buffer&*	2
10x T4 Milli Q H₂O	15.5
pDNA**	2
Restriction enzymes***	0.25

Results
plate	# of colonies	Components (µL)
1	200µL xylE-dt July 12	0
2	200µL mms6-dt July 12	1
3	200µL lumazine-dt	1
4	200µL mms6 maxiprep July 6	Lawn
5	200µL positve control puC19 into BL21(DE3)	Lawn
6	200µL xylE-dt July 14	0
7	200µL mms6-dt July 14	120
8	200µL lumazine-dt July14	0
9	200µL mms6 maxiprep July 10	.

Component	Volume (µL)
Milli-Q H₂O<	54
Pfu Buffer + MgSO₄	20
10 mM dMTPs	5
forward primer	5
reverse primer	5

Component	Volume (µL)
Milli-Q H₂O	15.5
Red Buffer₄	2
SpeI	0.25
EcoRI	0.25
pDNA	2

Lane	Content	Quantity of DNA (ng/µL)
1	1kB Ladder	12.5
3	pLacI restriction digest	5.21
5	sRBS restriction digest	3.72
7	xylErestriction digest	3.36
9	Lumazine restriction digest	2.63
11	dT restriction digest	2.98

Cell Pellet	Weight (g)
ECFP	2.32
EYFP	2.00
Lumazine Synthase	2.66