Team:UT Dallas/notebook data

June 6

Single and Double Population Projects

Transformation

• BBa_J61002: “RFP” promoter
• BBa_K081012: GFP protein generator (RBS-GFP)
• BBa_J23103: constitutive family member
• Bba_J04450: RFP Coding Device (pLas-RBS-RFP-Term)

Single and Double Population Projects

June 7

Colonies picked and glycerol stocked made

• RFP promoter: 5 colonies seen: 1 colony picked
• RBS-GFP: 2 colonies seen: 1 colony picked
• constitutive family promoter: 2 colonies seen: 1 colony picked
• pLas-RBS-RFP-Term: 6 colonies seen: 1 colony picked

Single and Double Population Projects

June 8

Plasmid isolation

• RBS-GFP
• Concentration:111.2 ng/ul
• constututive family member
• Concentration: 109.8 ng/ul
• pLasI-RBS-RFP-Term
• Concentration: 113.9 ng/ul

Single and Double Population Projects

June 11

Digestion

• RBS-GFP (E/S)---(wrong enzymes used)
• constitutive promoter (E/X)---(wrong enzymes used)
• constitutive promoter (S/P)
• pLasI-RBS-RFP-Term (X/P)

Single and Double Population Projects

June 12

Digestion

• RBS-GFP (X/P)

Ligation

• Vector: constitutive promoter
• Insert: RBS-GFP
• 0:1--- 0 colonies counted
• 2:1--- 7 colonies counted

June 13

Incubate constitutive promoter-RBS-GFP in LB carb broth overnight @ 37*C and 220 rpm

• 2:1--- 2 colonies picked

Single and Double Population Projects

June 14

Glycerol stock and miniprep

• constitutive promoter-RBS-GFP
• colony 1 picked: 91 ng/ul
• colony 2 picked: 97 ng/ul

Test Digestion for each plasmid: correct results received

Single and Double Population Projects

June 15

Plasmid isolation

• prom-RBS-GFP: Concentration: 127 ng/µl

Single and Double Population Projects

July 16

Transformations

• Bba_I763035 pTetR-RBS-LacI-RBS-GFP-Term-Term
• Bba_K093005 RBS-RFP
• Bba_R0010 pLacI
• Bba_C0040 TetR

Single and Double Population Projects

July 17

• Bba_I763035 pTetR-RBS-LacI-RBS-GFP-Term-Term
• 2 colonies picked
• Bba_K093005 RBS-RFP
• 2 colonies picked
• Bba_R0010 pLacI
• 2 colonies picked
• Bba_C0040 TetR
• 2 colonies picked

Single and Double Population Projects

July 18

Glycerol stocks made

• pTetR-RBS-LacI-RBS-GFP-Term-Term
• Concentration: 236.2 ng/µl
• RBS-RFP
• Concentration: 92.9 ng/µl
• pLacI
• Concentration: 60.6 ng/µl
• TetR
• Concentration: 110.9 ng/µl

Digestion

• pTetR-RBS-LacI-RBS-GFP-Term-Term (E/P)
• RBS-RFP (X/P)
• pLacI (S/P)
• TetR (X/P)

Gel Extraction

• RBS-RFP
• Concentration: 10.8 ng/µl
• pLac
• Concentration: 19.6 ng/µl
• TetR
• Concentration: 6.2 ng/µl

Ligation

• Vector: pLac
• Insert: TetR

Transformation

• pLac-TetR
• 0:1, 0 colonies seen
• 2:1, 20 colonies seen, 1 picked
• 4:1, 20 colonies seen, 1 picked

July 19

Triple Population and AND Gate Projects

Transformations

• BBa_I721001: lead promoter
• BBa_K091109: LuxS
• BBa_C0078: LasI
• BBa_C0062: LuxR
• BBa_C0061: LuxI
• BBa_E0432: RBS-YFP

July 20

Triple Population and AND Gate Projects

Colonies picked

• LuxS: 20 colonies seen: 1 colony picked
• LuxR: 5 colonies seen: 1 colony picked
• LuxI: 5 colonies seen: 1 colony picked
• RBS-YFP: no colonies seen

July 20

Single and Double Population Projects

Glycerol stocks & plasmid isolation

• pLas-TetR: Concentrations: (2:1) 67.4 and (4:1) 68.0

Digestion

• (2:1) pLac-TetR

Gel Extraction

• pLac-TetR: Concentration: 20.5 ng/µl

Ligation

• Vector: pLac-TetR
• Insert: RBS-YFP

Transformation

• pLac-TetR-RBS-YFP
• (0:1) no colonies seen

Single and Double Population Projects

Triple Population and AND Gate Projects

July 20

Repeat Transformation

• lead promoter
• LasI
• RBS-YFP

Triple Population and AND Gate Projects

July 21

Glycerol Stock

• LuxS
• LuxR
• LuxI

Colonies picked

• RBS-YFP: 1 colony picked

Digestion & Gel Extraction

• LuxS (wrong enzymes used)
• LuxR: Concentration: 11.1 ng/µl
• LuxI: Concentration: 7.1 ng/µl

Triple Population and AND Gate Projects

July 22

Plasmid Isolation

• LuxS: Concentration: 75.2 ng/µl
• LasI: Concentration: 90.1 ng/µl
• RBS-YFP: Concentration: 76.4 ng/µl

Triple Population and AND Gate Projects

July 23

DIgestion and gel extraction

• LuxS (X/P) Concentration: not right size to be repeated
• LasI (E/S) Concentration: 20.3 ng/µl
• RBS-YFP (X/P) Concentration: 18.2 ng/µl
• (wrong enzymes used in digestions - plasmid isolation repeated)

Ligation

• Vector: pLac
• Insert: LuxR

Transformation

• BBa_ K117002: pLsrA

Colonies picked

• pLac-LuxR: 15 colonies seen: 1 colony picked
• pLsrA: 20 colonies seen: colony picked
• pLuxR: 3 colonies seen: 1 colony picked

July 23

Glycerol stock and plasmid isolation

• pLac-TetR-RBS-YFP: Concetration: 138.4

Digestion

• pLac-TetR-RBS-YFP (X/P)
• pTetR-LacI-RBS-GFP (S/P)

Ligation

• Vector: pTetR-LacI-RBS-GFP
• Insert: pLac-TetR-RBS-YFP
• 2 colonies picked after plating and incubation

Triple Population and AND Gate Projects

July 24

Plasmid Isolation

• LuxS: Concentration: 85.9 ng/µl
• LuxI: Concentration:85.7 ng/µl
• LasI: Concentration: 224.1 ng/µl

Digestion and gel extraction

• LuxI (S/P): Concentration: 25.5 ng/µl
• LasI (S/P): Concentration: 30.7 ng/µl

Ligation

• Vector 1: LuxI
• Insert 1: RBS-YFP
• Vector 2: LasI
• Insert 2: RBS-RFP

Triple Population and AND Gate Projects

July 24

Transformation

• BBa_C0079: LasR
• BBa_B0015: Term-Term
• BBa_I14016: pLac
• BBa_C0012: LacI

Triple Population and AND Gate Projects

July 25

Colonies picked

• LuxI-RBS-YFP: 100 colonies seen: 1 picked
• LasI-RBS-RFP: 50 colonies seen: 1 picked

Plasmid Isolation

• pLac-LuxR: Concentration: 107.5
• pLsrA: Concentration: 44.3
• pLuxR: Concentration: 35.9

Digestion and gel extraction

• pLsrA (S/P): Concentration: 20.6 ng/µl

Transformation

• BBa_R0040: pTetR
• BBa_R0079: pLasR
• BBa_K17036: RBS-CFP

Single and Double Population Projects

July 25

Glycerol Stock and plasmid isolation

• pTetR-LacI-RBS-GFP-pLac-TetR-RBS-YFP (full toggle switch) from both colines picked
• Concentration 1: 194.8 ng/µl
• Concentartion 2: 117.2 ng/µl

Transformation

• BBa_C0079: LasR
• BBa_I14016: pLas
• BBa_C0012: LacI

Tetracycle Stock: 50 ng/?l

IPTG stock: 50 ng/?l

Triple Population and AND Gate Projects

July 26

Colonies picked

• pTetR: 10 colonies seen: 1 colony picked
• pLasR: 10 colonies seen: 1 colony picked
• RBS-CFP: no colonies seen

Glycerol stock and plasmid isolation

• LuxI-RBS-YFP: Concentration: 70.8 ng/µl
• LasI-RBS-RFP: Concentration: 4.0 (to be repeated) ng/µl

Digestion and gel extraction

• LuxI-RBS-YFP (X/P): Concentration: 2.8 (to be repeated)

Single and Double Population Projects

July 26

Transformation

• BBa_C0012: LacI

Triple Population and AND Gate Projects

July 27

Glycerol stock and plasmid isolation

• pTetR: Concentration: 42.4 ng/µl
• pLasR: Concentration: 62.2 ng/µl
• LuxI-RBS-YFP: Concentration: 120.9 ng/µl

• LasI-RBS-RFP: Concentration: 85 ng/µl

Digestion and gel extraction

• pTetR (S/P): Concentration: 17.4 ng/µl
• pLasR (S/P): Concentration: 23.8 ng/µl
• LuxI-RBS-YFP (X/P): Concentration: 13.8 ng/µl

• LasI-RBS-RFP (X/P): Concentration: 8.9 ng/µl

Ligation

• Vector 1: pLsrA
• Insert 1:LuxI-RBS-YFP

Transformation:

• pLsrA-LuxI-RBS-YFP
• pLuxR-LasI-RBS-RFP

• BBa_B0422: RBS-CFP

Single and Double Population Projects

July 27

Colonies picked

• LacI: 8 colonies seen: 1 colony picked
• LacI (2): 2 colonies seen: 1 colony picked

Plasmid isolation

• LasR: Concentration: 95.8 ng/µl
• Term-Term: Concentration: 120.1 ng/µl

Toggle switch test

• IPTG innoculation

Triple Population and AND Gate Projects

July 28

Colonies picked

• pLsrA-LuxI-RBS-YFP: 20 colonies seen: 1 colony picked
• pLuxR-LasI-RBS-RFP: 2 colonies seen: 1 colony picked
• RBS-CFP: 10 colonies seen: 1 colony picked

Single and Double Population Projects

July 28

Glycerol stock and plasmid isolation

• LacI: Concentration: 73.9 ng/µl
• LacI (2): Concentration: 109.8 ng/µl

Triple Population and AND Gate Projects

July 29

Glycerol Stocks

• pLsrA-LuxI-RBS-YFP: Concentration: 201.6 ng/µl
• pLuxR-LasI-RBS-RFP: Concentration: 127.1 ng/µl
• RBS-CFP: Concentration: 84.0 ng/µl

Triple Population and AND Gate Projects

July 30

Plasmid Isolation

• pLsrA-LuxI-RBS-YFP: Concentration: 201.6 ng/µl
• pLuxR-LasI-RBS-RFP: Concentration: 127.1 ng/µl
• RBS-CFP: Concentration: 84.0 ng/µl

Digestion and gel extraction

• LuxS (X/P): Gel extraction failed
• pLuxR-LasI-RBS-RFP (X/P): Gel extraction failed
• RBS-CFP (X/P): Concentration: 10.4 ng/µl
• pLac-LuxR (S/P): Concentration: 21.0 ng/µl

Innoculation

• pLuxR-LasI-RBS-RFP
• LuxS

Ligation and transformation:

• Vector: pLasR
• Insert: RBS-CFP
• 2 colonies shown: 1 colony picked

Single and Double Population Projects

July 30

Experiment 7

• 150 ng/?l Tet
• 200 ng/?l Tet
• 300 ng/?l Tet
• 1mM IPTG
• No Induction

Innoculation

• RBS-GFP

Triple Population and AND Gate Projects

July 31

Plasmid isolation

• LuxS: Concentration: 78.1 ng/µl
• pLuxR-LasI-RBS-RFP: Concentration: 48.7 ng/µl

Digestion:

• LuxS: (X/P); 516 bp

• pLuxR+LasI-RBS-RFP: Digested at XbaI and PstI; 1421 bp

Test Digestion:

• LuxS: digested at EcoRI and PstI

• pLuxR-lasI-RBS-RFP: digested at EcoRI and PstI

Colonies:

• pLuxR-lasI-RBS-RFP: 1 additional colony picked

• LuxS: 1 additional colony picked

Single and Double Population Projects

July 31

Plasmid Isolation:

• GFP: 137.5 ng/µl

August 1, 2012:

Plasmid Isolation:

• pLuxR-LasI-RBS-RFP: 61.2 ng/µl

• pLasR-RBS-CFP: 49.2 ng/µl

Digestion:

• GFP: Digested XbaI and PstI

• LacI: Digested XbaI and PstI

• pLuxR-LasI-RBS-RFP: Digested XbaI and PstI

• pLusR-RBS-CFP: Digested XbaI and PstI

Gel Exraction:

• GFP: 8.5 ng/µl

• LacI: 12.7 ng/µl

Colony:

• pLasR-RBS-CFP: 1 additional colony picked

Transformation:

• pLuxR-lasI-RBS-RFP

• BBa_J2309: pTetR-luxI

Single and Double Population Projects

August 1

Ligation:

• Vector: pTet; Insert: GFP

• Vector: pTet; Insert: LacI

• Vector: pLac; Insert: GFP

Transformation:

• pTet-GFP

• pTet-LacI

• pLac-GFP

Triple Population and AND Gate Projects

August 2, 2012:

Colonies:

• pLuxR-LasI-RBS-RFP: about 5 colonies; 1 picked

• pTetR-LuxI: about 2 colonies; 1 picked

Plasmid Isolation:

• LuxS: Concentration – 40.5 ng/µl

• pLasR-CFP: 95.8 ng/µl

Digestion:

• LasI: Digested at XbaI and PstI

• LasR: Digested at XbaI and PstI

• LuxS: Digested at XbaI and PstI

• pLasR-RBS-CFP: Digested at XbaI and PstI

Gel Extraction:

• LasI: Concentration – 4.1 ng/µl

• LasR: Concentration – 5.3 ng/µl

• pLasR-RBS-CFP – 13.4 ng/µl

Ligation:

• Vector: pLac

• Insert: LasI

• Transformation 2:1 – 3 colonies; 1 picked

Single and Double Population Projects

August 2

Colonies:

• pTet-GFP: 7 colonies; 1 picked

• pTet-LacI: 20 colonies; 1 picked

• pLac-GFP: 20 colonies; 1 picked

Single and Double Population Projects

August 3, 2012:

Glycerol Stocks/Miniprep:

• pTet-GFP: Concentration-142.2 ng/µl

• pTet-LacI: Concentration-67.2 ng/µl

• pLac-GFP: Concentration-50.5 ng/µl

Digestion:

• pTet-GFP: Digested at SpeI and PstI

• pTet-LacI: Digested at SpeI and PstI

• pLac-GFP: Digested at XbaI and PstI

• pLac-TetR: Digested at XbaI and PstI

Triple Population and AND Gate Projects

August 3

Ligation:

• Vector: pTetR

• Insert: LasR

• Transformation 2:1 – 1 colony; 1 picked

Ligation:

• Vector: pLasR

• Insert: RBS-CFP

• Transformation: 2:1

Glycerol Stock:

• pLuxR-LasI-RBS-RFP: Concentration – 45.1 ng/µl

• pTetR-LuxI: Concentration – 105.1 ng/µl

Digestion:

• pLuxR-LasI-RBS-RFP: Digested at XbaI and PstI

Ligation:

• Vector: pLac-luxR
• Insert: pLuxR-LasI-RBS-RFP

Transformation

• BBa_K091109 (2010) LuxS
• BBa_K091109 (2011) LuxS

Triple Population and AND Gate Projects

August 4

Colonies picked

• BBa_K091109 (2010) LuxS: 70 colonies seen: 1 picked
• BBa_K091109 (2011) LuxS: 100 colonies seen: 1 picked

Triple Population and AND Gate Projects

August 5

Glycerol Stock

• LuxS
• LuxS
• pTetR-RBS-CFP

Transformation (repeated)

• pLasR-RBS-CFP

Triple Population and AND Gate Projects

August 6

Colonies picked

• pLasR-RBS-CFP: 10 colonies seen: 1 colony picked

Innoculation

• pTetR-LuxI

Plasmid isolation

• LuxS: Concentration: 57.6

Digestion and gel extraction (improperly prepared for gel)

• LuxS: Concentration: N/A
• LuxS: Concentration: N/A
• pTetR-RBS-CFP: Concentration: 21.9

Innoculation

• LuxS
• LuxS
• pTetR-RBS-CFP

Single and Double Population Projects

August 6, 2012:

PCR:

RFP-LVA created

Experiments:

Test mechanism shows TetR and LacI are medium strength repressors.

Toggle Experiment fails to show significant changes in fluorescence protein after 2 or 6 hours.

Triple Population and AND Gate Projects

August 7

Glycerol Stock

• pLasR-RBS-CFP

Plasmid isolation

• LuxS: Concentration: 120.3
• LuxS: Concentration:101.7
• pTetR-LasR: Concentration: 65.3
• pLasR-RBS-CFP: Concentration: 77.1
• pLac-luxR-pLuxR-LasI-RBS-RFP: Concentration: 103.6

Digestion and gel extraction

• LuxS (X/P): Concentration: 1.5
• LuxS (X/P): Concentration: 1.1
• pTetR-LasR (S/P): Concentration: 20.4

Ligation

• Vector 1: pTetR-lasR
• Insert 1: pLasR-RBS-CFP
• Vector 2: pyear
• Insert 2: LuxS

Transformation

• pSBIC3 chlora backbone
• pLac-luxR-pLuxR-LasI-RBS-RFP

Single and Double Population Projects

August 7, 2012

Glycerol Stock/Miniprep

pTet-LacI

pLac-GFP

Digestion & Gel Extraction

pTet-LacI: XbaI and PstI

pLac-GFP: SpeI and PstI

Ligation/Transformation

Vector: pLac-GFP

Insert: pTet-LacI

Triple Population and AND Gate Projects

August 8

Colonies picked

• chlora bb: 100 colonies seen: 1 picked
• pLac-luxR-pLuxR-LasI-RBS-RFP: 300 colonies seen: 1 picked
• pTetR-lasR-pLasR-RBS-CFP: 1 colony seen: 1 picked

Repeat transformations from ligation

• pyear-LuxS
• pTetR-lasR-pLasR-RBS-CFP

Single and Double Population Projects

August 8, 2012

Transformations:

Negative control had ~20 colonies

2:1 sample had ~ 40 colonies, we picked 2

Positive had too many colonies to count

PCR Reaction (Gradient)

-55, 60, and 65 degrees

-55 degrees found to be most ideal

Gel Extraction (of PCR)

Poor results, no DNA extracted

Plate test for toggle switch:

1 tube inoculated with IPTG 10 microliters of .2mM solution

1 tube inoculated with ATC 10 microliters of 20ng/microliter

Triple Population and AND Gate Projects

August 9

Glycerol stocks

• chlora backbone
• pLac-LuxR-pLuxR-LasI-RBS-RFP

Innoculation

• pLac-Las-I
• pTetR-LuxI

Plasmid Isolation

• chlora backbone

• pLac-LuxR-pLuxR-LasI-RBS-RFP
• pTetR-LasR-pLasR-RBS-CFP

Digestion

• Term-Term (S/P)

• chlora backbone (E/P)

• pLac-LuxR-pLuxR-LasI-RBS-RFP (E/P)
• pTetR-LasR-pLasR-RBS-CFP (E/P)

Gel Extraction

• Term-Term: Concentration: 44.3

• chlora backbone: Concentration: 332.9

• pLac-LuxR-pLuxR-LasI-RBS-RFP: bad results
• pTetR-LasR-pLasR-RBS-CFP: bad results

Innoculation: pLac-LuxR-pLuxR-LasI-RBS-RFP

Single and Double Population Projects

August 9, 2012

Glycerol stock/miniprep:

pLac-GFP-ST-pTet-LacI

PCR Reaction

RFP-LVA

Experiment: Repressor Test Mechanism (LacI)

1 - Induced 10microliters of 10mM IPTG

1 - No induction

Digestion and Gel Extraction:

RFP-LVA: XbaI and PstI

Ampicillin Backbone: SpeI and PstI

August 10

Glycerol Stock

• pLac-LuxR-pLuxR-LasI-RBS-RFP

Plasmid isolation

• pLac-LuxR-pLuxR-LasI-RBS-RFP: Concentration: 125.1

Digestion

• pLac-LuxR-pLuxR-LasI-RBS-RFP (E/P)

Ligation

• Vector1: Term-Term
• Insert1: pLasR-RBS-CFP
• Vector2: Term-Term
• Insert2: pLuxR-LasI-RBS-RFP

Transformation:

• Term-Term-pLasR-RBS-CFP
• Term-Term-pLuxR-LasI-RBS-RFP

Single and Double Population Projects

August 10, 2012

Ligation/Transformation

RFP-LVA into Ampicillin backbone

Inoculations:

Toggle

pLas

LacI

LuxR

pTet

pLux

LuxI

August 11, 2012

Plasmid isolation

Toggle

pLas

LacI

LuxR

pTet

pLux

LuxI

Digestion/Extraction

LuxR: X and P

pLux: S and P

LuxI: S and P

LasR: X and P

Double Terminators (DT): X and P

LasI: X and P

Triple Population and AND Gate Projects

August 11

Colonies picked

• Term-Term-pLasR-RBS-CFP: 200 colonies seen: 1 picked
• Term-Term-pLuxR-LasI-RBS-RFP: 100 colonies seen: 1 picked

Triple Population and AND Gate Projects

August 12

Glycerol Stock and Plasmid isolation

• Term-Term-pLasR-RBS-CFP: Concentration: 219.1
• Term-Term-pLuxR-LasI-RBS-RFP: Concentration: 107.5

Transformation repeat

• pyear-LuxS

Digestion

• Term-Term-pLasR-RBS-CFP (X/P)
• Term-Term-pLuxR-LasI-RBS-RFP (X/P)

Gel Extraction

• Term-Term-pLasR-RBS-CFP: Concentration: 10.2
• Term-Term-pLuxR-LasI-RBS-RFP: Concentration: 15.0

Ligation

• Vector1: pTetR-LasR
• Insert1: Term-Term-pLasR-RBS-CFP

Triple Population and AND Gate Projects

August 13

Innoculation

• pLac-LasI
• pTetR-LuxI

Single and Double Population Projects

August 13, 2012

Miniprep/Glycerol Stock

RFP-LVA

Digestions/Extraction

pLas: S and P

LacI: X and P

RFP-LVA: E and P

Inoculations

Chlorabackbone

RFP-LVA

DT

Triple Population and AND Gate Projects

August 14

Glycerol Stock and Plasmid isolation

• pTetR-LasR-Term-Term-pLasR-RBS-CFP: Concentration: 95.4
• pLac-LuxR-Term-Term-pLuxR-LasI-RBS-RFP: Concentration: 108.3

Digestion

• pTetR-LasR-Term-Term-pLasR-RBS-CFP: bad results
• pLac-LuxR-Term-Term-pLuxR-LasI-RBS-RFP: bad results

Parts Recieved

• BBa_K091159
• BBa_K091002
• BBa_I13453
• BBa_B0034

Streaking

• BBa_K091159 pBad-LsrA

Ligation repeat

• Vector1: pTetR-LasR
• Insert1: Term-Term-pLasR-RBS-CFP
• Vector2: pLac-LuxR
• Insert 2: Term-Term-pLuxR-LasI-RBS-RFP

Single and Double Population Projects

August 14, 2012

Miniprep/Glycerol Stock

Chlorabackbone

DT

Digestion/Extraction:

DT: X and P

Chlorabackbone: E and P

Toggle: E and P

Ligation/Transformation:

DT into LuxR

TetR into pLux

LasI into pTet

LasR into DT

pLas into LacI

pLac into LuxI

Triple Population and AND Gate Projects

August 15

Innoculation

• pTetR-LuxI
• pLac-LasI

• pTetR-LasR-Term-Term-pLasR-RBS-CFP
• pLac-LuxR-Term-Term-pLuxR-LasI-RBS-RFP

Colonies picked

• pBad-LsrA: 30 colonies seen: 1 picked

Testing

• Pop2 (w/o TT) / pTetR-LuxI (48 hours of incubation)
• 100/0 no results
• 100/100 no results
• 100/200 slight RFP detected no increase of RFP from 100/0
• Pop3 (w/o TT) / pLac-LasI (48 hours of incubation)
• 100/0 no results
• 100/100 no results
• 100/200 slight RFP detected no increase of CFP from 100/0

Streaking

BBa_K091109

• SP3011>1D LuxS
• SP4011>1D LuxS

Single and Double Population Projects

August 15, 2012

Transformation

LuxR-DT: 15 colonies, picked 1 colony

pLux-TetR: 35 colonies, picked 1

pTet-LasI: Negative control growth; unsuccessful

LasR-DT: 25 colonies, picked 1

pLas-LacI: 0 growth

pLac-LuxI: 15 colonies, picked 1

Ligation

LasI into pTet

Transformation

pTet-LasI

pLas-LacI

Inoculations

Chlorabackbone

Triple Population and AND Gate Projects

August 16

Colonies

• pTetR-LuxI:
• pLac-LasI

• pTetR-LasR-Term-Term-pLasR-RBS-CFP: 2 colonies seen: 1 colony picked
• pLac-LuxR-Term-Term-pLuxR-LasI-RBS-RFP: 0 colonies seen
• LuxS: 200 colonies seen: 1 colony picked
• LuxS: 100 colonies seen: 1 colony picked

Glycerol Stock and plasmid isolation

• pBad-LsrK: Concentration: 64.2
• pTetR-LasR-Term-Term-pLasR-RBS-CFP: Concentration:
• pLac-LuxR-Term-Term-pLuxR-LasI-RBS-RFP: Concentration:

Digestion

• pTetR-LasR-Term-Term-pLasR-RBS-CFP (E/P)
• pLac-LuxR-Term-Term-pLuxR-LasI-RBS-RFP (E/P)
• pBad-LsrA (S/P) 1749
• pLsrA-LuxI-RBS-YFP (X/P)

Testing

• Pop2 (w/ TT) / pTetR-LuxI (6 hours of incubation)
• 100/0
• 100/200
• 100/400
• 100/600
• Results: After 6 and 24 hours: no considerable difference
• Pop3 (w/ TT) / pLac-LasI (6 hours of incubation)
• 100/0
• 100/200
• 100/400
• 100/600
• Results: After 6 and 24 hours: no considerable difference
• Pop3 (w/ TT) / Pop2 (w/ TT) / pTetR-LuxI (6 hours of incubation)
• 100/0
• 100/200

Digestion

• population 3 (oscillator)
• population 2 (oscillator)
• pBad->lsrK
• pLsrA-LuxI-RBS-YFP
• Term-Term (test)

Gel Extraction

• pBad-lsrK: Concentration: 53.8
• pLsrA-LuxI-RBS-YFP: Concentration: 20.0

Innoculation

• Term-term

Single and Double Population Projects

August 16, 2012

Miniprep/Glycerol Stock

LuxR-DT

pLux-TetR

LasR-DT

pLac-LacI

Chlorabackbone

Transformation Results

Negative controls had too much growth; results thrown out

Digestion/Extraction

LuxR-DT: S and P

pLux-TetR: X and P

Chlorabackbone: E and P

Ligations/Transformation

LuxR-DT and pLux-TetR

Chlorabackbone with Toggle Switch

Triple Population and AND Gate Projects

August 17

Glycerol Stock

• Population 3 w/ Term-Term
• LuxS
• LuxS

Plasmid Isolation

• Population 3 w/ Term-Term: Concentration: 126.7
• LuxS: Concentration: 40.2
• LuxS: Concentration: 52.4
• Term-Term: Concentration: 100.8

Digestion and Gel Extraction

• Population 3 w/ Term-Term: Concentration: gel not used
• LuxS: Concentration: gel not used
• LuxS: Concentration: gel not used

Ligation

• Vector 1: pLac
• Insert 1: lasI-RBS-RFP
• Vector 2: pLasR
• Insert 2: LuxI-RBS-YFP
• Vector 3: pbad-LsrK
• Insert 3: pLsrA->YFP
• Vector 4: pLac->LuxR
• Insert 4: pLuxR-RFP
• Vector 5: pTetR-LasR
• Insert 5: pLasR->CFP

Transformation

• BBa_C0080: araC
• BBa_J24674: pBad-TetR
• BBa_I14015: pLas-TetO

Single and Double Population Projects

August 17, 2012

Ligation

pLas with LacI

pTet with LasI

Inoculations

pLas

LacI

pTet

pLac

GFP

RFP-LVA

LasI

Transformation

Bba_K081014 Kit Plate 3 Well 12O Resistance AK; RFP

Triple Population and AND Gate Projects

August 18

Innoculations

• pTetR-LuxI
• pLac-lasI
• pop 2 w/TT (oscillator)
• pop 3 w/TT (oscillator)

Single and Double Population Projects

August 18, 2012

Colony Picking

LuxR-DT-pLux-TetR: 30 colonies, 1 picked

Toggle Switch in Chlorabackbone: 4 colonies, 1 picked

pLas with LacI: negative control growth; results thrown out

pTet with LasI: negative control growth; results thrown out

RFP: 20 colonies, 1 picked

Glycerol Stock

LuxR-DT-pLux-TetR

Minipreps

LuxR-DT-pLux-TetR

pLas

LacI

pTet

pLac

GFP

LasI

Triple Population and AND Gate Projects

August 19

Glycerol Stocks

• pLac-lasI-RBS-RFP
• pLasR-LuxI-RBS-YFP
• pBad-LsrK-plsrA-LuxI-RBS-YFP
• plac-LuxR-pLuxR-LasI-RBS-RFP
• pTetR-lasR-pLasR-RBS-CFP
• araC
• pBad-TetR
• pLas-TetO

Oscillator Population Tests (population with induction plasmid)

• pop2 0:1 - some fluorescence
• pop2 75:1 - no visual difference
• pop3 0:1 - some fluorescence
• pop3 75:1 - no visual difference
• pop2 and pop3 0:1 - little fluorescence

Plasmid Isolation

• pLac-lasI-RBS-RFP: Concentration: 115.8
• pLasR-LuxI-RBS-YFP: Concentration: 93.1
• pBad-LsrK-plsrA-LuxI-RBS-YFP: Concentration: 128.6
• plac-LuxR-pLuxR-LasI-RBS-RFP: Concentration: 105.9
• pTetR-lasR-pLasR-RBS-CFP: Concentration: 122.3
• araC: Concentration: 114.0
• pBad-TetR: Concentration: 315.1
• pLas-TetO: Concentration: 102.3

Digestion and gel extraction

• ad-LsrK-plsrA-LuxI-RBS-YFP: Concentration:
• plac-LuxR-pLuxR-LasI-RBS-RFP: Concentration:
• pTetR-lasR-pLasR-RBS-CFP: Concentration:
• araC: Concentration:
• pBad-TetR: Concentration:

Ligation

• Vector 1: pyear
• Insert 1: LuxS
• Vector 2: pyear
• Insert 2: LuxS
• Vector 3: pTetR
• Insert 3:lasR-TT
• Vector 4: pLac-LuxR
• Insert 4: TT
• Vector 5: pLuxR
• Insert 5: arac
• Vector 6: pBad-TetR

Single and Double Population Projects

August 19, 2012

Glycerol Stock/Plasmid Isolation

RFP

Toggle Switch in Chlorabackbone

Inoculation

RFP-LVA

Digestion/Extraction

pLac: S and P

LuxR-DT-pLux-TetR: X and P

pTet: S and P

LasI: X and P

LasR-DT: X and P

LacI: S and P

RFP: X and P

Triple Population and AND Gate Projects

August 20

Colonies picked

• pyear-LuxR: 1 picked
• pyear-LuxR: 1 picked
• pTet-lasR-TT: 1 picked
• pLac-LuxR-TT: 1 picked
• pBad-TetR-TT: 1 picked
• pLuxR-arac: 1 picked
• pLasR-TetO-RBS-CFP: 1 picked after being repeated

Innoculation

• pTetR-LuxI
• pLac-lasI
• pop3 w/TT

Single and Double Population Projects

August 20, 2012

Ligations/Transformations

LuxR-DT-pLux-TetR into pLac

LasI into pTet

LasR-DT into pTet

RFP into LacI

Triple Population and AND Gate Projects

August 21

Glycerol Stock and plasmid isolation

• pyear-LuxR: Concentration: 40.0
• pyear-LuxR: Concentration: 75.0
• pTet-lasR-T: Concentration: 127.6

Digestion and gel extraction

• pLasR-LuxI-YFP: gel not used
• pTetR-lasR-TT: gel not used
• pLuxR-araC: gel not used
• pyear-LuxS: gel not used
• pyear-LuxS: gel not used
• pop1 (test)
• pop2 (test)
• pop3 (test)

Innoculation

• pyear-LuxS

Ligation

• Vector 1: pTet-LasR-TT

Single and Double Population Projects

August 21, 2012

Colony Pickings

pLac-LuxR-DT-pLux-TetR: 24 colonies, 1 picked

pTet-LasI: 30 colonies, 3 picked

pTet-LasR-DT: 100 colonies, 2 picked

LacI-RFP: 24 colonies, 2 picked

Triple Population and AND Gate Projects

August 22

Digestion (test)

• population 2 (oscillator)
• undigested
• EcoRI

Innoculation

• population 1 (oscillator)

Colonies picked

• pTetR-lasR-TT-pLasR-LuxI-RBS-YFP
• pLuxR-araC-LasR-TT
• pLasR-TetO-RBS-CFP (2 colonies picked)

Transformation

• pLac-LuxR

Innoculation

• pLuxR-LasI-RBS-RFP

Population Testing Prep (pop2, pop3, pop2+pop3)

• 2 ml of medium added to each test tube
• 100 microliters of each population added as necessary
• .75mL of inducing population added as necessary
• pop2 w/ w/o inducer
• pop3 w/ w/o inducer
• pop2+pop3 w/ w/o inducer

Single and Double Population Projects

August 22, 2012

Glycerol Stock/Miniprep

pLac-LuxR-DT-pLux-TetR

pTet-LasI

pTet-LasR-DT

LacI-RFP

Digestion/Extraction:

pLac-LuxR-DT-pLux-TetR: S and P

GFP: X and P

LacI-RFP: S and P

pLac-LuxI: X and P

Test Digestions

pTet-LasI: E and P

pTet-LasR-DT: E and P

Ligations/Transformations

GFP into pLac-LuxR-DT-pLux-TetR

pLac-LuxI into LacI-RFP

Inoculations

RFP-LVA

pTet-GFP

RBS-YFP

August 22

Digestion (test)

• population 2 (oscillator)

• undigested
• EcoRI
• XbaI
• Spe1
• Pst1
• E/P
• X/S

Innoculation

• population 1 (oscillator)

Colonies picked

• pTetR-lasR-TT-pLasR-LuxI-RBS-YFP
• pLuxR-araC-LasR-TT
• pLasR-TetO-RBS-CFP (2 colonies picked)

Transformation

• pLac-LuxR

Innoculation

• pLuxR-LasI-RBS-RFP

Population Testing Prep (pop2, pop3, pop2+pop3)

• 2 ml of medium added to each test tube
• 100 microliters of each population added as necessary
• .75mL of inducing population added as necessary
• pop2 w/ w/o inducer
• pop3 w/ w/o inducer
• pop2+pop3 w/ w/o inducer

Single and Double Population Projects

August 23, 2012

Colony Picking

LacI-RFP-ST-pLac-LuxI; 40 colonies, 1 picked

LacI-RFP-ST-pLac-LuxI; 30 colonies, 1 picked

Triple Population and AND Gate Projects

August 23

Glycerol Stock

• pLasR-TetO-RBS-CFP (X2)
• pTetR-lasR-TT-pLasR-LuxI-RBS-YFP
• pLuxR-araC-LasR-TT

Plasmid Isolation

• pLasR-TetO-RBS-CFP: Concentration: 113.7
• pLasR-TetO-RBS-CFP: Concentration: 112.0

• pTetR-lasR-TT-pLasR-LuxI-RBS-YFP: Concentration: 167.0
• pLuxR-araC-LasR-TT: Concentration: 104.2
• pLac-lasI-RBS-RFP: Concentration: 37.0

Colonies picked

• pLac-LuxR

Population Testing from 8/22 prep after 24 hours

• 500 microliters from test tube of populations put in microcentrifuge tubes

• pop2 w/: slight increase of RFP
• pop2 w/o: some RFP
• pop3 w/: no significant increase of CFP

• test tubes were checked after 24 hours
• pop2 showed a slight increase in RFP production when it was induced in comparison to its uninduced control
• both pop3 and pop2+pop3 showed no significant differences between its positive and negative controls
• after 48 hours
• pop2 showed increase in RFP production
• pop3 showed no significant increase in CFP production
• pop2+pop3 showed a sig increase in RFP production but no CFP when it was induced compared to its negative control

Digestion

• plac-LuxR(S/P): Concentration: 19.7

Innoculation

• pyear-luxS
• pBad-LsrK-pLsaA-LuxI-RBS-YFP (pop1 oscillator)
• pTetR-LuxI
• pLacI-LuxR-TT-pLuxR-LasI-RBS-RFP (pop2 oscillator)
• pLac-lasI
• pTetR-lasR-TT-pLasR-RBS-CFP (pop3 oscillator)

Ligation

• Vector: pBad-TetR-TT
• Insert: plasR-TetO-RBS-CFP

Single and Double Population Projects

August 24, 2012

Plasmid Isolation

LacI-RFP-ST-pLac-LuxI

Digestion/Extraction

pTet-LasR-DT

pTet-LasI

LacI-RFP-ST-pLac-LuxI

pLas

Ligation

pTet-LasI into pLac-LuxR-DT-pLux-TetR-GFP-ST

Triple Population and AND Gate Projects