Team:UT Dallas/notebook data

July 19

Transformations

● BBa_I721001: lead promoter

● BBa_K091109: LuxS

● BBa_C0078: LasI

● BBa_C0062: LuxR

● BBa_C0061: LuxI

● BBa_E0432: RBS-YFP

July 20

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

● LasI: no colonies seen

● lead promoter: no colonies seen

July 20

Repeat Transformation

● lead promoter

● LasI

● RBS-YFP

July 21

Glycerol Stock

● LuxS

● LuxR

● LuxI

Colonies picked

● RBS-YFP: 1 colony picked

● LasI: 1 colony picked

● lead promoter: no colones seen

Digestion & Gel Extraction

● LuxS (wrong enzymes used)

● LuxR: Concentration: 11.1 ng/μl

● LuxI: Concentration: 7.1 ng/μl

July 22

Plasmid Isolation

● LuxS: Concentration: 75.2 ng/μl

● LasI: Concentration: 90.1 ng/μl

● RBS-YFP: Concentration: 76.4 ng/μl

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

● BBa_R0062: pLuxR

● BBa_I721001: lead promoter

Colonies picked

● pLac-LuxR: 15 colonies seen: 1 colony picked

● pLsrA: 20 colonies seen: colony picked

● pLuxR: 3 colonies seen: 1 colony picked

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

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

● pLuxR (S/P): Concentration: 18.7 ng/μl

Transformation

● BBa_R0040: pTetR

● BBa_R0079: pLasR

● BBa_K17036: RBS-CFP

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)

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

● Vector 2: pLuxR

● Insert 2:LasI-RBS-RFP

Transformation:

● pLsrA-LuxI-RBS-YFP

● pLuxR-LasI-RBS-RFP

● BBa_B0422: RBS-CFP

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

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

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

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

August 1

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 Extraction:

• 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

August 2

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

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

August 4

Colonies picked

● BBa_K091109 (2010) LuxS: 70 colonies seen: 1 picked

● BBa_K091109 (2011) LuxS: 100 colonies seen: 1 picked

August 5

Glycerol Stock

● LuxS

● LuxS

● pTetR-RBS-CFP

Transformation (repeated)

● pLasR-RBS-CFP

August 6

Colonies picked

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

Innoculation

● pTetR-LuxI

Plasmid isolation

● LuxS: Concentration: 57.6

● LuxS: Concentration: 76.4

● pTetR-RBS-CFP: Concentration: 122.3

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

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

● pLasR-RBS-CFP (X/P): Concentration: 13.8

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

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

August 9

Glycerol stocks

● chlora backbone

● pLac-LuxR-pLuxR-LasI-RBS-RFP

● pTetR-LasR-pLasR-RBS-CFP

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

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

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

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

● Vector2: pLac-LuxR

● Insert 2: Term-Term-pLuxR-LasI-RBS-RFP

August 13

Innoculation

● pLac-LasI

● pTetR-LuxI

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

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

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

○ 100/400

○ 100/600

○ Results: After 6 and 24 hours: no considerable difference

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

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

● Term-Term: 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

August 18

Innoculations

● pTetR-LuxI

● pLac-lasI

● pop 2 w/TT (oscillator)

● pop 3 w/TT (oscillator)

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

● pop2 and pop3 75:1 - a visual increase of both RFP and CFP

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: gel not used

● plac-LuxR-pLuxR-LasI-RBS-RFP: gel not used

● pTetR-lasR-pLasR-RBS-CFP: gel not used

● araC: gel not used

● pBad-TetR: gel not used

● pLas-TetO: gel not used

● LasR-TT: Concentration: 10.0

● LuxS: Concentration: 3.1

● LuxS: Concentration: 5.5

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

● Insert 6: TT

● Vector 7: plasR-TetO

● Insert 7: RBS-CFP

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

● pop2 w/TT

● pop3 w/o TT

● pop2 w/o TT

August 21

Glycerol Stock and plasmid isolation

● pyear-LuxR: Concentration: 40.0

● pyear-LuxR: Concentration: 75.0

● pTet-lasR-T: Concentration: 127.6

● pLac-LuxR-TT: Concentration: 123.5

● pBad-TetR-TT: Concentration: 141.0

● pLuxR-arac: Concentration: 84.0

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

● Insert 1: pLasR-LuxI-RBS-YFP

● Vector 2: pLuxR-araC

● Insert 2: lasR-TT

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

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

○ pop3 w/o: some CFP

○ pop2+pop3 w/: no significant increase of RFP/CFP

○ pop2+pop3 w/o: some RFP/CFP

● Results:

○ 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

● pTetR-LasR (E/P): Concentration: gel not used

● pLasR-RBS-CFP (X/P): Concentration: gel not used

● pLac-LuxR-TT (S/P): Concentration: 24.0

● pBad-TetR-TT (S/P): Concentration: 15.4

● pTetR-LasR-TT-pLasR-LuxI-RBS-YFP (E/P): Concentration: 12.2

● pLuxR-araC-lasR-TT (X/P): Concentration: gel not used

● pLasR-TetO-RBS-CFP (X/P): Concentration: 5.2

● pLasR-TetO-RBS-CFP (X/P): Concentration: 5.2

● pLuxR-LasI-RBS-RFP (X/P): Concentration: gel not used

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)

● pLuxR-lasI-RBS-RFP

Ligation

● Vector: pBad-TetR-TT

● Insert: plasR-TetO-RBS-CFP

August 24

Plasmid Isolation

● pluxR-LasI-RBS-RFP: Concentration: 42.6

Digestion

● pLuxR-LasI-RBS-RFP (X/P)

● pLuxR-araC-lasR-TT (X/P)

Innoculation

● pLuxR-LasI-RBS-RFP

● pLuxR-araC-LasR-TT

● pLsrA-LuxI-YFP

August 25

Glycerol Stock

● plac-LuxR

● pBad-TetR-TT-plasR-TetO-RBS-CFP

● pBad-TetR-TT-plasR-TetO-RBS-CFP

● pLuxR-araC-lasR-TT

Plasmid Isolation

● pLuxR-LasI-RBS-RFP: Concentration: 89.1

● pLuxR-araC-LasR-TT: Concentration: 152.9

● pBad-TetR-TT-plasR-TetO-RBS-CFP: Concentration: 200.0

● pBad-TetR-TT-plasR-TetO-RBS-CFP: Concentration: 156.0

Digestion and Gel Extraction

● pLuxR-LasI-RBS-RFP: (X/P): Concentration: 9.5

● pLuxR-araC-LasR-TT: (X/P): gel not used

● pBad-TetR-TT-plasR-TetO-RBS-CFP: (X/P): gel not used

● pBad-TetR-TT-plasR-TetO-RBS-CFP: (X/P): gel not used

Ligation

● Vector1: pLac-LuxR-TT

● Insert1: pLuxR-LasI-RBS-RFP

● Vector2: pTetR-LasR

● Insert2: pLasR-CFP

● Vector3: pLac-LuxR-TT

● Insert3: pLuxR-LasI-RBS-RFP

Primer Dilution

● I3.PsB1A2.R +661ul NFH2O -> 1:10 Dulition

● I3.DT.F +634ul NFH2O -> 1:10 Dulition

● I3.FT.M +700ul NFH2O -> 1:10 Dulition

Colony PCR

● pBad-TetR-TT-plasR-TetO-RBS-CFP

● pLuxR-araC-LasR-TT

○ Primer 1: I3.24.SP1

○ Primer 2: I3.PSB1A2.R

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)

● pLuxR-lasI-RBS-RFP

August 26

Colonies Picked

● pLuxR-araC-LasR-TT: 1 colony picked

Population testing

● 2ml broth, 1ml induction, 100ul population, 30 hour incubation

○ pop1+2 (+/-): no fluorescence seen

○ pop2+3 (+/-): no fluorescence seen

○ pop1+2+3 (+/-): no fluorescence seen

August 27

Plasmid Isolation

● pLuxR-araC-LaR-TT: Concentration: 164.3

Digestion and Gel extraction

● pLuxR-araC-LaR-TT (X/P): Concentration: 55.1

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)

● pLuxR-lasI-RBS-RFP

Ligation

● Vector1: pBad-tetR-TT

● Insert1: pLasR-TetO-RBS-CFP

○ 0:1 20 colonies seen

○ 2:1 50 colonies seen

● Vector2:pLac-LuxR-TT

● Insert2: pLuXR-araC-lasR-TT

○ 0:1 5 colonies seen

○ 2:1 25 colonies seen

August 28

Colony PCR

● pBad-tetR-TT-pLasR-TetO-RBS-CFP

● pLac-LuxR-TT- pLuXR-araC-lasR-TT

○ Primer 1: I3.24.SP1

○ Primer 2: I3.PSB1A2.R

Population Testing

● 200ul medium, 200ul induction, 4ul population, 24 hour incubation

○ pop1+2 (-): no fluorescence seen

○ pop1+2 (+): some sign of RFP/YFP

○ pop2+3 (-): no fluorescence seen

○ pop2+3 (+): no fluorescence seen

○ pop1+2+3 (-): no fluorescence seen

○ pop1+2+3 (+): no fluorescence seen

Ligation

● Vector:pLac-LuxR-TT

● Insert: pLuXR-araC-lasR-TT

○ 0:1 0 colonies seen

○ 2:1 30 colonies seen

○ 4:1 15 colonies seen

Digestion and Gel Extraction

● pBad-TetR-TT (S/P): Concentration: 88.3

Innoculation

● pyear-luxS

● pBad-LsrK-pLsaA-LuxI-RBS-YFP (pop1 oscillator)

● pTetR-LuxI

● pLacI-LuxR-TT-pLuxR-LasI-RBS-RFP (pop2 oscillator)

● pTetR-lasR-TT-pLasR-RBS-CFP (pop3 oscillator)

August 29

Population Test Prep

● 2ml medium, 400ul populations, 1500ul induction

○ pop1+2 (+/-)

○ pop2+3 (+/-)

○ pop1+2+3 (+/-)

● 1ml medium, 200ul populations, 750ul induction

○ pop2+3 (+/-)

August 30

Ligation

● Vector: pBad-TetR-TT

● Insert: plasR-TetO-CFP

○ 0:1 100 Colonies seen

○ 1:1 200 Colonies seen

○ 4:1 200 Colonies seen

Colony PCR

● pLac-LuxR-TT-pLuXR-araC-lasR-TT (X2)

○ Primer 1: I3.24.SP1

○ Primer 2: I3.PSB1A2.R

Colonies picked

● pLac-LuxR-TT-pLuXR-araC-lasR-TT (X2)