Team:UT Dallas/notebook data

From 2012.igem.org

(Difference between revisions)
 
Line 222: Line 222:
<ul>
<ul>
     <li>
     <li>
-
         prom-RBS-GFP: Concentration: 127 ng/μl
+
         prom-RBS-GFP: Concentration: 127 ng/µl
     </li>
     </li>
</ul>
</ul>
Line 320: Line 320:
     <ul>
     <ul>
         <li>
         <li>
-
             Concentration: 236.2 ng/μl
+
             Concentration: 236.2 ng/µl
         </li>
         </li>
     </ul>
     </ul>
Line 328: Line 328:
     <ul>
     <ul>
         <li>
         <li>
-
             Concentration: 92.9 ng/μl
+
             Concentration: 92.9 ng/µl
         </li>
         </li>
     </ul>
     </ul>
Line 336: Line 336:
     <ul>
     <ul>
         <li>
         <li>
-
             Concentration: 60.6 ng/μl
+
             Concentration: 60.6 ng/µl
         </li>
         </li>
     </ul>
     </ul>
Line 344: Line 344:
     <ul>
     <ul>
         <li>
         <li>
-
             Concentration: 110.9 ng/μl
+
             Concentration: 110.9 ng/µl
         </li>
         </li>
     </ul>
     </ul>
Line 380: Line 380:
     <ul>
     <ul>
         <li>
         <li>
-
             Concentration: 10.8 ng/μl
+
             Concentration: 10.8 ng/µl
         </li>
         </li>
     </ul>
     </ul>
Line 388: Line 388:
     <ul>
     <ul>
         <li>
         <li>
-
             Concentration: 19.6 ng/μl
+
             Concentration: 19.6 ng/µl
         </li>
         </li>
     </ul>
     </ul>
Line 396: Line 396:
     <ul>
     <ul>
         <li>
         <li>
-
             Concentration: 6.2 ng/μl
+
             Concentration: 6.2 ng/µl
         </li>
         </li>
     </ul>
     </ul>
Line 544: Line 544:
<ul>
<ul>
     <li>
     <li>
-
         pLac-TetR: Concentration: 20.5 ng/μl
+
         pLac-TetR: Concentration: 20.5 ng/µl
     </li>
     </li>
</ul>
</ul>
Line 661: Line 661:
     </li>
     </li>
     <li>
     <li>
-
         LuxR: Concentration: 11.1 ng/μl
+
         LuxR: Concentration: 11.1 ng/µl
     </li>
     </li>
     <li>
     <li>
-
         LuxI: Concentration: 7.1 ng/μl
+
         LuxI: Concentration: 7.1 ng/µl
     </li>
     </li>
</ul>
</ul>
Line 687: Line 687:
<ul>
<ul>
     <li>
     <li>
-
         LuxS: Concentration: 75.2 ng/μl
+
         LuxS: Concentration: 75.2 ng/µl
     </li>
     </li>
     <li>
     <li>
-
         LasI: Concentration: 90.1 ng/μl
+
         LasI: Concentration: 90.1 ng/µl
     </li>
     </li>
     <li>
     <li>
-
         RBS-YFP: Concentration: 76.4 ng/μl
+
         RBS-YFP: Concentration: 76.4 ng/µl
     </li>
     </li>
</ul>
</ul>
Line 717: Line 717:
     </li>
     </li>
     <li>
     <li>
-
         LasI (E/S) Concentration: 20.3 ng/μl
+
         LasI (E/S) Concentration: 20.3 ng/µl
     </li>
     </li>
     <li>
     <li>
-
         RBS-YFP (X/P) Concentration: 18.2 ng/μl
+
         RBS-YFP (X/P) Concentration: 18.2 ng/µl
     </li>
     </li>
     <li>
     <li>
Line 835: Line 835:
<ul>
<ul>
     <li>
     <li>
-
         LuxS: Concentration: 85.9 ng/μl
+
         LuxS: Concentration: 85.9 ng/µl
     </li>
     </li>
     <li>
     <li>
-
         LuxI: Concentration:85.7 ng/μl
+
         LuxI: Concentration:85.7 ng/µl
     </li>
     </li>
     <li>
     <li>
-
         LasI: Concentration: 224.1 ng/μl
+
         LasI: Concentration: 224.1 ng/µl
     </li>
     </li>
</ul>
</ul>
Line 850: Line 850:
<ul>
<ul>
     <li>
     <li>
-
         LuxI (S/P): Concentration: 25.5 ng/μl
+
         LuxI (S/P): Concentration: 25.5 ng/µl
     </li>
     </li>
     <li>
     <li>
-
         LasI (S/P): Concentration: 30.7 ng/μl
+
         LasI (S/P): Concentration: 30.7 ng/µl
     </li>
     </li>
</ul>
</ul>
Line 948: Line 948:
<ul>
<ul>
     <li>
     <li>
-
         pLsrA (S/P): Concentration: 20.6 ng/μl
+
         pLsrA (S/P): Concentration: 20.6 ng/µl
     </li>
     </li>
Line 955: Line 955:
     <li>
     <li>
-
         pLuxR (S/P): Concentration: 18.7 ng/μl
+
         pLuxR (S/P): Concentration: 18.7 ng/µl
     </li>
     </li>
</ul>
</ul>
Line 992: Line 992:
     <ul>
     <ul>
         <li>
         <li>
-
             Concentration 1: 194.8 ng/μl
+
             Concentration 1: 194.8 ng/µl
         </li>
         </li>
         <li>
         <li>
-
             Concentartion 2: 117.2 ng/μl
+
             Concentartion 2: 117.2 ng/µl
         </li>
         </li>
     </ul>
     </ul>
Line 1,015: Line 1,015:
</ul>
</ul>
<p>
<p>
-
     Tetracycle Stock: 50 ng/чl
+
     Tetracycle Stock: 50 ng/?l
</p>
</p>
Line 1,022: Line 1,022:
<div id="page18" class='notebook_page'>
<div id="page18" class='notebook_page'>
<p>
<p>
-
     IPTG stock: 50 ng/чl
+
     IPTG stock: 50 ng/?l
</p>
</p>
Line 1,055: Line 1,055:
<ul>
<ul>
     <li>
     <li>
-
         LuxI-RBS-YFP: Concentration: 70.8 ng/μl
+
         LuxI-RBS-YFP: Concentration: 70.8 ng/µl
     </li>
     </li>
     <li>
     <li>
-
         LasI-RBS-RFP: Concentration: 4.0 (to be repeated) ng/μl
+
         LasI-RBS-RFP: Concentration: 4.0 (to be repeated) ng/µl
     </li>
     </li>
</ul>
</ul>
Line 1,111: Line 1,111:
<ul>
<ul>
     <li>
     <li>
-
         pTetR: Concentration: 42.4 ng/μl
+
         pTetR: Concentration: 42.4 ng/µl
     </li>
     </li>
     <li>
     <li>
-
         pLasR: Concentration: 62.2 ng/μl
+
         pLasR: Concentration: 62.2 ng/µl
     </li>
     </li>
     <li>
     <li>
-
         LuxI-RBS-YFP: Concentration: 120.9 ng/μl
+
         LuxI-RBS-YFP: Concentration: 120.9 ng/µl
     </li>
     </li>
</ul>
</ul>
Line 1,123: Line 1,123:
<ul>
<ul>
     <li>
     <li>
-
         LasI-RBS-RFP: Concentration: 85 ng/μl
+
         LasI-RBS-RFP: Concentration: 85 ng/µl
     </li>
     </li>
</ul>
</ul>
Line 1,132: Line 1,132:
<ul>
<ul>
     <li>
     <li>
-
         pTetR (S/P): Concentration: 17.4 ng/μl
+
         pTetR (S/P): Concentration: 17.4 ng/µl
     </li>
     </li>
     <li>
     <li>
-
         pLasR (S/P): Concentration: 23.8 ng/μl
+
         pLasR (S/P): Concentration: 23.8 ng/µl
     </li>
     </li>
     <li>
     <li>
-
         LuxI-RBS-YFP (X/P): Concentration: 13.8 ng/μl
+
         LuxI-RBS-YFP (X/P): Concentration: 13.8 ng/µl
     </li>
     </li>
</ul>
</ul>
Line 1,144: Line 1,144:
<ul>
<ul>
     <li>
     <li>
-
         LasI-RBS-RFP (X/P): Concentration: 8.9 ng/μl
+
         LasI-RBS-RFP (X/P): Concentration: 8.9 ng/µl
     </li>
     </li>
</ul>
</ul>
Line 1,216: Line 1,216:
<ul>
<ul>
     <li>
     <li>
-
         LasR: Concentration: 95.8 ng/μl
+
         LasR: Concentration: 95.8 ng/µl
     </li>
     </li>
     <li>
     <li>
-
         Term-Term: Concentration: 120.1 ng/μl
+
         Term-Term: Concentration: 120.1 ng/µl
     </li>
     </li>
</ul>
</ul>
Line 1,281: Line 1,281:
<ul>
<ul>
     <li>
     <li>
-
         LacI: Concentration: 73.9 ng/μl
+
         LacI: Concentration: 73.9 ng/µl
     </li>
     </li>
     <li>
     <li>
-
         LacI (2): Concentration: 109.8 ng/μl
+
         LacI (2): Concentration: 109.8 ng/µl
     </li>
     </li>
</ul>
</ul>
Line 1,305: Line 1,305:
<ul>
<ul>
     <li>
     <li>
-
         pLsrA-LuxI-RBS-YFP: Concentration: 201.6 ng/μl
+
         pLsrA-LuxI-RBS-YFP: Concentration: 201.6 ng/µl
     </li>
     </li>
     <li>
     <li>
-
         pLuxR-LasI-RBS-RFP: Concentration: 127.1 ng/μl
+
         pLuxR-LasI-RBS-RFP: Concentration: 127.1 ng/µl
     </li>
     </li>
     <li>
     <li>
-
         RBS-CFP: Concentration: 84.0 ng/μl
+
         RBS-CFP: Concentration: 84.0 ng/µl
     </li>
     </li>
</ul>
</ul>
Line 1,329: Line 1,329:
<ul>
<ul>
     <li>
     <li>
-
         pLsrA-LuxI-RBS-YFP: Concentration: 201.6 ng/μl
+
         pLsrA-LuxI-RBS-YFP: Concentration: 201.6 ng/µl
     </li>
     </li>
     <li>
     <li>
-
         pLuxR-LasI-RBS-RFP: Concentration: 127.1 ng/μl
+
         pLuxR-LasI-RBS-RFP: Concentration: 127.1 ng/µl
     </li>
     </li>
     <li>
     <li>
-
         RBS-CFP: Concentration: 84.0 ng/μl
+
         RBS-CFP: Concentration: 84.0 ng/µl
     </li>
     </li>
</ul>
</ul>
Line 1,350: Line 1,350:
     </li>
     </li>
     <li>
     <li>
-
         RBS-CFP (X/P): Concentration: 10.4 ng/μl
+
         RBS-CFP (X/P): Concentration: 10.4 ng/µl
     </li>
     </li>
     <li>
     <li>
-
         pLac-LuxR (S/P): Concentration: 21.0 ng/μl
+
         pLac-LuxR (S/P): Concentration: 21.0 ng/µl
     </li>
     </li>
</ul>
</ul>
Line 1,405: Line 1,405:
<ul>
<ul>
     <li>
     <li>
-
         150 ng/чl Tet
+
         150 ng/?l Tet
     </li>
     </li>
     <li>
     <li>
-
         200 ng/чl Tet
+
         200 ng/?l Tet
     </li>
     </li>
     <li>
     <li>
-
         300 ng/чl Tet
+
         300 ng/?l Tet
     </li>
     </li>
     <li>
     <li>
Line 1,447: Line 1,447:
<ul>
<ul>
     <li>
     <li>
-
         LuxS: Concentration: 78.1 ng/μl
+
         LuxS: Concentration: 78.1 ng/µl
     </li>
     </li>
     <li>
     <li>
-
         pLuxR-LasI-RBS-RFP: Concentration: 48.7 ng/μl
+
         pLuxR-LasI-RBS-RFP: Concentration: 48.7 ng/µl
     </li>
     </li>
</ul>
</ul>
Line 1,519: Line 1,519:
<ul>
<ul>
     <li>
     <li>
-
         GFP: 137.5 ng/μl
+
         GFP: 137.5 ng/µl
     </li>
     </li>
</ul>
</ul>
Line 1,535: Line 1,535:
<ul>
<ul>
     <li>
     <li>
-
         pLuxR-LasI-RBS-RFP: 61.2 ng/μl
+
         pLuxR-LasI-RBS-RFP: 61.2 ng/µl
     </li>
     </li>
</ul>
</ul>
Line 1,541: Line 1,541:
<ul>
<ul>
     <li>
     <li>
-
         pLasR-RBS-CFP: 49.2 ng/μl
+
         pLasR-RBS-CFP: 49.2 ng/µl
     </li>
     </li>
</ul>
</ul>
Line 1,580: Line 1,580:
<ul>
<ul>
     <li>
     <li>
-
         GFP: 8.5 ng/μl
+
         GFP: 8.5 ng/µl
     </li>
     </li>
</ul>
</ul>
Line 1,586: Line 1,586:
<ul>
<ul>
     <li>
     <li>
-
         LacI: 12.7 ng/μl
+
         LacI: 12.7 ng/µl
     </li>
     </li>
</ul>
</ul>
Line 1,708: Line 1,708:
<ul>
<ul>
     <li>
     <li>
-
         LuxS: Concentration – 40.5 ng/μl
+
         LuxS: Concentration – 40.5 ng/µl
     </li>
     </li>
</ul>
</ul>
Line 1,714: Line 1,714:
<ul>
<ul>
     <li>
     <li>
-
         pLasR-CFP: 95.8 ng/μl
+
         pLasR-CFP: 95.8 ng/µl
     </li>
     </li>
</ul>
</ul>
Line 1,752: Line 1,752:
<ul>
<ul>
     <li>
     <li>
-
         LasI: Concentration – 4.1 ng/μl
+
         LasI: Concentration – 4.1 ng/µl
     </li>
     </li>
</ul>
</ul>
Line 1,758: Line 1,758:
<ul>
<ul>
     <li>
     <li>
-
         LasR: Concentration – 5.3 ng/μl
+
         LasR: Concentration – 5.3 ng/µl
     </li>
     </li>
Line 1,767: Line 1,767:
<ul>
<ul>
     <li>
     <li>
-
         pLasR-RBS-CFP – 13.4 ng/μl
+
         pLasR-RBS-CFP – 13.4 ng/µl
     </li>
     </li>
</ul>
</ul>
Line 1,842: Line 1,842:
<ul>
<ul>
     <li>
     <li>
-
         pTet-GFP: Concentration-142.2 ng/μl
+
         pTet-GFP: Concentration-142.2 ng/µl
     </li>
     </li>
</ul>
</ul>
Line 1,848: Line 1,848:
<ul>
<ul>
     <li>
     <li>
-
         pTet-LacI: Concentration-67.2 ng/μl
+
         pTet-LacI: Concentration-67.2 ng/µl
     </li>
     </li>
</div>
</div>
Line 1,857: Line 1,857:
<ul>
<ul>
     <li>
     <li>
-
         pLac-GFP: Concentration-50.5 ng/μl
+
         pLac-GFP: Concentration-50.5 ng/µl
     </li>
     </li>
</ul>
</ul>
Line 1,951: Line 1,951:
<ul>
<ul>
     <li>
     <li>
-
         pLuxR-LasI-RBS-RFP: Concentration – 45.1 ng/μl
+
         pLuxR-LasI-RBS-RFP: Concentration – 45.1 ng/µl
     </li>
     </li>
</ul>
</ul>
Line 1,957: Line 1,957:
<ul>
<ul>
     <li>
     <li>
-
         pTetR-LuxI: Concentration – 105.1 ng/μl
+
         pTetR-LuxI: Concentration – 105.1 ng/µl
     </li>
     </li>
</ul>
</ul>
Line 7,640: Line 7,640:
     </li>
     </li>
</ul>
</ul>
-
 
</div>
</div>
Line 7,688: Line 7,687:
     </li>
     </li>
</ul>
</ul>
 +
</div>
</html>
</html>

Latest revision as of 01:28, 4 October 2012

Cover page

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
  • LasI: no colonies seen
  • lead promoter: 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
  • (2:1) 1 colony picked

    • 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
  • 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

    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
  • 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 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
  • pLuxR (S/P): Concentration: 18.7 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
  • BBa_I732100: LacI (2)

    • 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
  • Vector 2: pLuxR
  • Insert 2:LasI-RBS-RFP

    • 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
  • TET innoculation
  • No 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
  • 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

    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
  • 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

    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
  • 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

    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

    Triple Population and AND Gate Projects

    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
  • Vector2: pLac-LuxR
  • Insert 2: Term-Term-pLuxR-LasI-RBS-RFP

    • 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
  • 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

    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
  • 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

    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
  • 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: Concentration:
    • plac-LuxR-pLuxR-LasI-RBS-RFP: Concentration:
    • pTetR-lasR-pLasR-RBS-CFP: Concentration:
    • araC: Concentration:
    • pBad-TetR: Concentration:
  • pLas-TetO: Concentration:
  • 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

    • 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
  • pop2 w/TT
  • pop3 w/o TT
  • pop2 w/o 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
  • 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

    • 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
  • 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 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
  • 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

    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

    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

    Single and Double Population Projects

    August 25, 2012

    Glycerol Stock/Plasmid Isolation

    LacI-RFP-ST-pLac-LuxI

    Digestion/Extraction

    LacI-RFP-ST-pLac-LuxI

    Ligation

    LacI-RFP-ST-pLac-LuxI into pLas

    Triple Population and AND Gate Projects

    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

    Triple Population and AND Gate Projects

    August 26, 2012

    Colony Picking

    pLas-LacI-RFP-ST-pLac-LuxI; 50 colonies, 1 picked

    Inoculation

    pTet-LacI-RBS-GFP(LVA)-T-T-pLac-TetR-RBS-RFP

    pLas-LacI-RFP-ST-pLac-LuxI

    Triple Population and AND Gate Projects

    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

    Single and Double Population Projects

    August 27, 2012

    Glycerol Stock/Plasmid Isolation

    pTet-LacI-RBS-GFP(LVA)-T-T-pLac-TetR-RBS-RFP

    pLas-LacI-RFP-ST-pLac-LuxI

    PCR

    Add second LacO site to pLac

    Digestion/Extraction

    pTet-LacI-RBS-GFP(LVA)-T-T-pLac-TetR-RBS-RFP

    pLas-LacI-RFP-ST-pLac-LuxI

    Triple Population and AND Gate Projects

    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

    Single and Double Population Projects

    August 28, 2012

    Primer Dilutions

    Triple Population and AND Gate Projects

    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)

    • Single and Double Population Projects

    August 29, 2012

    Ligation

    pLas-LacI-RFP-ST-pLac-LuxI into pTet-LasR-DT

    Triple Population and AND Gate Projects

    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 (+/-)

    Triple Population and AND Gate Projects

    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)

    Single and Double Population Projects

    August 31, 2012

    Colony picking

    pTet-LasR-DT-pLas-LacI-RFP-ST-pLac-LuxI; 50 colonies, 1 picked

    PCR Reaction

    Add LacO binding site to pLac; 65.2 degrees found to be optimal temperature for elongation

    Transformation

    pLac-LacO

    Triple Population and AND Gate Projects

    August 31

    Glycerol Stock and Plasmid Isolation

    • pLac-LuxR-TT-pLuXR-araC-lasR-TT: Concentration: 115.2
    • pLac-LuxR-TT-pLuXR-araC-lasR-TT: Concentration: 77.7

    Digestion and Gel Extraction

    • pLac-LuxR-TT-pLuXR-araC-lasR-TT (S/P)
  • pLac-LuxR-TT-pLuXR-araC-lasR-TT (S/P)

    • Colony PCR

    • pBad-TetR-TT-pLasR-TetO-CFP (X2)
    • Primer 1: I3.24.SP1
    • Primer 2: I3.PSB1A2.R

    Population Test Results

    • 2ml medium, 400ul populations, 1500ul induction
      • pop1+2 (+/-)
      • pop2+3 (+/-)
      • pop1+2+3 (+/-)
    • 1ml medium, 200ul populations, 750ul induction
      • pop2+3 (+/-)

    Single and Double Population Projects

    September 1, 2012

    Glycerol Stock/Plasmid Isolation

    pTet-LasR-DT-pLas-LacI-RFP-ST-pLac-LuxI

    Colony Picking

    pLac-LacO

    Inoculations

    pLac-TetR

    pLac-TetR-RBS-RFP

    pTet-LacI

    pTet-RBS-GFP-T

    Triple Population and AND Gate Projects

    September 1

    Ligations

    • Vector1: pLac-LuxR-TT
    • Insert1: pLuxR-araC-LasR-TT
    • Vector2: pLasR
    • Insert2.1: LuxS
    • Insert2.2 araC
    • Vector3: TT
  • Insert3: pLsrA-LuxI-RBS-YFP
  • Vector4: pLuxR
  • Insert4: TetR

    • Transformation

    • BBa_C0012: LacI
    • BBa_K091002: LsrK

    Innoculation

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

    Single and Double Population Projects

    September 2, 2012

    Glycerol Stock/Plasmid Isolation

    pLac-LacO

    Single and Double Population Projects

    September 3, 2012

    Plasmid Isolation

    pLac-TetR

    pLac-TetR-RBS-RFP

    pTet-LacI

    pTet-RBS-GFP-T

    Digestion/Extraction

    pLac-TetR

    pLac-TetR-RBS-RFP

    pTet-LacI

    pTet-RBS-GFP-T

    Triple Population and AND Gate Projects

    September 3

    Glycerol Stock

    • pLac-LuxR-TT-pLuxR-araC-LasR-TT
    • pBad-TetR-TT-pLas-TetO-CFP

    Plasmid Isolation

    • pLacI-LuxR-TT-pLuxR-LasI-RBS-RFP (pop2 oscillator)
    • pLac-LuxR-TT-pLuxR-araC-lasR-TT
    • pBad-TetR-TT-pLas-TetO-CFP

    Digestion

    • pLacI-LuxR-TT-pLuxR-LasI-RBS-RFP (pop2 oscillator) (X/P)
    • pBad-TetR-TT-pLas-TetO-CFP (X/P)
    • pLac-LuxR-TT-pLuxR-araC-lasR-TT (S/P)

    Innoculation

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

    • Colony PCR for I3-98-2 and I4-1-2-

    Digestion

    • TT-pLuxR-LasI-RFP (X/P)
    • pLac-LuxR (S/P)

    Single and Double Population Projects

    September 4, 2012

    Inoculations

    pLac-LuxR-DT-pLux-TetR-GFP-ST-pTet-LasI (Atc 10 microliters)

    pTet-LasR-DT-pLas-LacI-RFP-ST-pLac-LuxI (IPTG 10 microliters)

    Triple Population and AND Gate Projects

    September 4

    Plasmid Isolation

    • pBad-TetR-TT-pLasR-TetO-CFP: Concentration: 190.4

    Digestion

    • pBad-TetR-TT-pLasR-TetO-CFP (X/P): Concentration: 20.5

    Colonies picked from I4-1-2

    Single and Double Population Projects

    September 5, 2012

    Inoculations

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

    pTet-LasR-DT-pLas-LacI-RFP-ST-pLac-LuxI

    1 with Atc

    1 with IPTG

    1 with No induction

    Triple Population and AND Gate Projects

    September 5

    Glycerol Stock and Plasmid Isolation

    • pLasR-LuxS
    • pLasR-araC
    • TT-pLsrA-LuxI-RBS-YFP
  • pLuxR-TetR
  • LacI
  • LsrK

    • Digestion and Gel Extraction

    • pLasR-LuxS
    • pLasR-araC
    • TT-pLsrA-LuxI-RBS-YFP
    • pLuxR-TetR
    • LacI
    • LsrK

    Innoculation

    • pLac
    • LasI-RFP

    Single and Double Population Projects

    September 6, 2012

    Sequencing

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

    pTet-LasR-DT-pLas-LacI-RFP-ST-pLac-LuxI

    pLac-LacO

    RFP-LVA

    Inoculations

    Chlorabackbone

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

    pTet-LasR-DT-pLas-LacI-RFP-ST-pLac-LuxI

    1 with Atc

    1 with IPTG

    1 with No induction

    Digestion/Extraction

    Chlorabackbone

    Triple Population and AND Gate Projects

    September 6

    Plasmid Isolation and Glycerol Stock

    • pLac
    • LasI-RFP
    • pLac-LuxR-TT-pLuxR-araC-LasR-TT

    Digestion and Gel Extraction

    • pLac
    • LasI-RFP
    • pLac-LuxR-TT-pLuxR-araC-LasR-TT

    Ligation

    • Vector1:pLac-LuxR-TT-pLuxR-araC-LasR-TT
    • Insert1: pBad->>CFP
    • Vector2: pLsrA
    • Insert2: LacI
    • Vector3: pLasR-LuxS
  • Insert3: RBS-CFP
  • Vector4: Term-Term
  • Insert4: pLuxR-TetR

    • Single and Double Population Projects

    September 7, 2012

    Inoculation

    pTet-LacI-RBS-GFP(LVA)-T-T-pLac-TetR-RBS-RFP

    pLac-RBS-GFP-T

    pTet-RBS-GFP-T-pLac-TetR

    pLac-RBS-GFP-T-pTet-LacI

    RFP-LVA

    pLac-LacO

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

    pTet-LasI

    pLac-LuxR-DT-pLux-TetR

    LuxR-DT-pLux-TetR

    LuxR-DT

    pLux-TetR

    pTet-LasR-DT-pLas-LacI-RFP-ST-pLac-LuxI

    pLas-LacI-RFP-ST-pLac-LuxI

    LacI-RFP-ST-pLac-LuxI

    LacI-RFP-ST

    pLac-LuxI

    pTet-LasR-DT

    LasR-DT

    pLac-LacO

    pTet-LacI

    RBS-GFP-ST

    Triple Population and AND Gate Projects

    September 7

    Test Digestions

    • pLac
    • LasI-RFP
  • pLac-LuxR-TT-pLuxR-araC-LasR-TT

    • Innoculation

    • Term-Term

    Single and Double Population Projects

    September 8, 2012

    Time-Lapse Experiment to observe toggle switch

    Plasmid Isolation

    pTet-LacI-RBS-GFP(LVA)-T-T-pLac-TetR-RBS-RFP

    pLac-RBS-GFP-T

    pTet-RBS-GFP-T-pLac-TetR

    pLac-RBS-GFP-T-pTet-LacI

    RFP-LVA

    pLac-LacO

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

    pTet-LasI

    pLac-LuxR-DT-pLux-TetR

    LuxR-DT-pLux-TetR

    LuxR-DT

    pLux-TetR

    pTet-LasR-DT-pLas-LacI-RFP-ST-pLac-LuxI

    pLas-LacI-RFP-ST-pLac-LuxI

    LacI-RFP-ST-pLac-LuxI

    LacI-RFP-ST

    pLac-LuxI

    pTet-LasR-DT

    LasR-DT

    pTet-LacI

    GFP

    Ligations

    pLac-TetR into Chlora

    pLac-RBS-GFP-T into Chlora

    pTet-RBS-GFP-T into Chlora

    pTet-LacI into Chlora

    ]

    Digestion/Extraction

    GFP

    pLac-LacO

    pTet-LacI-RBS-GFP(LVA)-T-T-pLac-TetR-RBS-RFP

    pLac-RBS-GFP-T

    pTet-RBS-GFP-T-pLac-TetR

    pLac-RBS-GFP-T-pTet-LacI

    RFP-LVA

    pLac-LacO

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

    pTet-LasI

    pLac-LuxR-DT-pLux-TetR

    LuxR-DT-pLux-TetR

    LuxR-DT

    pLux-TetR

    pTet-LasR-DT-pLas-LacI-RFP-ST-pLac-LuxI

    pLas-LacI-RFP-ST-pLac-LuxI

    LacI-RFP-ST-pLac-LuxI

    LacI-RFP-ST

    pLac-LuxI

    Triple Population and AND Gate Projects

    September 8

    Colonies Picked

    • 14-7-5
    • 14-7-7
    • 14-8-2

    Single and Double Population Projects

    September 9, 2012

    Colony Picking

    pLac-TetR, 1 colony picked

    pLac-RBS-GFP-T, 1 colony picked

    pTet-LacI, 1 colony picked

    Ligation

    pLac-LacO-GFP

    Triple Population and AND Gate Projects

    September 9

    Glycerol Stock and Plasmid Isolation

    • pLac-LuxR-TT-pLuxR-araC-LasR-TT-pBad-TetR-TT-pLasR-TetO-CFP
    • pLsrA-LacI
    • pLasR-LuxS-RBS-CFP

    Digestion and Gel Extraction

    • pLac-LuxR-TT-pLuxR-araC-LasR-TT-pBad-TetR-TT-pLasR-TetO-CFP
  • pLsrA-LacI
  • pLasR-LuxS-RBS-CFP

    • Ligations

    • Vector1: Term-Term
    • Insert1-1: pLsrA-LacI
    • Insert1-2: pLuxR-TetR
    • Insert1-3: pLsrA-LuxI-RBS-YFP
    • Insert1-4: pLasR-LuxS-RBS-CFP
    • Insert1-5: pLasR-araC
    • Vector2: pLac
    • Insert2: 13-12-8

    Single and Double Population Projects

    September 10, 2012

    Glycerol Stock/Miniprep

    pLac-TetR

    pLac-RBS-GFP-T


    pTet-LacI

    Gel Extraction

    pLac-LacO-GFP

    pTet-LacI

    Ligation

    pTet-LacI into pLac-LacO-RBS-GFP-ST

    pLac-RBS-GFP-T-pTet-LacI into chlora backbone

    Triple Population and AND Gate Projects

    September 10

    Colonies Picked

    • I4-11-2
    • I4-11-3
    • I4-11-4
    • I4-11-5
    • I4-11-6
    • I4-8-4
  • I4-8-5
  • I4-8-6

    • Plasmid Isolation

    • pLac
    • LuxR

    Single and Double Population Projects

    September 11, 2012

    PCR

    pLac-LacO

    pLac-LacO x2

    Single and Double Population Projects

    September 12, 2012

    Digestion/Gel Extraction

    pTet-LacI-pLac-LacO-RBS-GFP-ST

    Experiments

    Toggle with 20 micro liters IPTG

    Toggle with 40 micro liters ATc

    Strain 1 of dual population toggle with inducer strain

    Strain 2 of dual population toggle with inducer strain

    Ligation

    pLac-LacO-RBS-GFP-ST-pTet-LacI into chlora backbone

    Triple Population and AND Gate

    September 13

    Glycerol Stock and Plasmid Isolation

    • I4-8-4
    • I4-8-5
    • I4-8-6
    • I4-11-2
    • I4-11-3
    • I4-11-4
    • I4-11-5
  • I4-11-6
  • I4-13-1
  • I4-13-2

    • 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: Const. Promoter
    • Insert1-1: I3-28-4
    • Insert1-2: I4-6-6
    • Insert1-3: I4-16-2
    • Vector2: TT-pLasR-LuxS-RBS-CFP
  • Insert2: TT-pLuxR-TetR
  • Vector3: pLac
  • Insert3: LuxR
  • Vector4: pLuxR
  • Insert4: LasI-RFP

    • Ligations for Chlora

    • I4-14-7
    • I4-14-8
    • I4-14-10

    Transformation

    • I4-18-1
    • I4-18-2
    • I4-18-3
    • I401804

    Innoculation

    • I3-28-2
  • I3-30-4
  • I3-11-1
  • I3-46-4

    • Single and Double Population Projects

    September 14, 2012

    Ligation

    pTet-LacI-RBS-GFP(LVA)-T-T-pLac-TetR-RBS-RFP into Chlora backbone

    pLac-RBS-GFP-T into Chlora backbone

    pTet-RBS-GFP-T-pLac-TetR into Chlora backbone

    pLac-RBS-GFP-T-pTet-LacI into Chlora backbone

    RFP-LVA into Chlora backbone

    pLac-LacO into Chlora backbone

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

    pTet-LasI into Chlora backbone

    pLac-LuxR-DT-pLux-TetR into Chlora backbone

    LuxR-DT-pLux-TetR into Chlora backbone

    LuxR-DT into Chlora backbone

    pLux-TetR into Chlora backbone

    pTet-LasR-DT-pLas-LacI-RFP-ST-pLac-LuxI into Chlora backbone

    pLas-LacI-RFP-ST-pLac-LuxI into Chlora backbone

    LacI-RFP-ST-pLac-LuxI into Chlora backbone

    LacI-RFP-ST into Chlora backbone

    pLac-LuxI into Chlora backbone

    pLac-LacO x2-RBS-GFP-ST-pTet-LacI

    Glycerol Stock/Plasmid Isolation

    pLac-LacO-GFP

    Digestion

    pLac-LacO-GFP

    pTet-LacI

    pLac-LacO x2-RBS-GFP-ST-pTet-LacI

    Inoculation

    pLac-GFP-pTet-LacI

    Triple Population and AND Gate

    September 15

    Plasmid Isolation

    • I4-16-4 CP-LasR
    • I4-16-5 CP-LsrK
    • I4-16-6 CP-LuxR
    • I4-17-4 TT-pLasR->>TetR
    • I4-17-6 pLac-LuxR
    • I4-17-8 pLuxR-LasI-RFP

    Colonies picked

    • I4-19-19
    • I4-22-8
    • I4-22-9
    • I4-21-2

    Digestion

    • I4-16-4
  • I4-16-5
  • I4-16-6
  • I4-17-4
  • I4-17-6
  • I4-17-8

    • Ligations

    • Vector1: pTetR-LasR
    • Insert1: TT-pLasR-LuxS-RFP-TT-pLuxR-TetR
    • Vector2: Term-Term
    • Insert2: pLac-LuxR
    • Vector3: pLuxR-LasII-RFP
    • Insert3: TT-pLsrA-LacI

    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)

    • Triple Population and AND Gate

    September 18

    Glycerol Stock and Plasmid Isolation

    • pTetR-LasR-TT-pLasR-LuxS-CFP-TT-pLuxR-TetR
    • TT-pLac-LuxR
    • pLuxR-LasI-RFP-TT-pLsrA-LacI

    Digestion and Gel Extraction

    • I4-10-7
    • I4-25-14
    • I4-25-16
    • I4-25-18

    Ligation

    • Vector1: CP-LasR
    • Insert1: TT
    • Vector2: TT-pLsrA-LuxI-YFP
    • Insert 2: TT-pLasR-araC
  • Vector3: TT
  • Insert3.1: pTetR->>TetR
  • Insert3.2: pLuxR->>LacI
  • Vector4: CP-LsrK
  • Insert4: TT-pLac-LuxR

    • Innoculation

    • Pop3 AND Gate
    • Pop2 AND Gate
    • Pop1 AND Gate

    Testing AND Gate

    • Pop2 and 3
    • Pop1 and 3
    • Pop 1 2 3

    Triple Population and AND Gate

    September 19

    Plate Experiments for AND Gate

    Triple Population and AND Gate

    September 20

    Glycerol Stock and Plasmid Isolation

    • Cp-LasR-TT
    • TT-pLsrA->>araC
    • TT-pTetR->>TetR
    • CP-LsrK->>LuxR
    • TT-pLuxR->>LacI

    Digestions (repeated 3X)

    • Cp-LasR-TT
    • TT-pLsrA->>araC
    • TT-pTetR->>TetR
    • CP-LsrK->>LuxR
    • TT-pLuxR->>LacI

    Triple Population and AND Gate

    September 22

    Ligations (Repeated)

    • Vector1: CP-LasR
    • Insert1: TT
    • Vector2: TT-pLsrA-LuxI-YFP
    • Insert 2: TT-pLasR-araC
    • Vector3: TT
    • Insert3.1: pTetR->>TetR
    • Insert3.2: pLuxR->>LacI
    • Vector4: CP-LsrK
    • Insert4: TT-pLac-LuxR

    Single and Double Population Projects

    September 22, 2012

    Single toggle experiment - No induction at start, check every 6 hours, induce with IPTG after first 6 hours, and induce with ATc in new tube after 12 Hours

    Triple Population and AND Gate

    September 22

    Ligations (Repeated)

    • Vector1: CP-LasR
    • Insert1: TT
    • Vector2: TT-pLsrA-LuxI-YFP
    • Insert 2: TT-pLasR-araC
    • Vector3: TT
    • Insert3.1: pTetR->>TetR
    • Insert3.2: pLuxR->>LacI
    • Vector4: CP-LsrK
    • Insert4: TT-pLac-LuxR

    Triple Population and AND Gate

    September 23

    Digestions

    • Cp-LasR-TT
    • TT-pLsrA->>araC
    • TT-pTetR->>TetR
    • CP-LsrK->>LuxR
    • TT-pLuxR->>LacI

    Innoculations for AND Gate

    • Pop1
    • Pop2
    • Pop3

    Testing AND Gate

    • Pop 1 3
    • Pop 2 3
    • Pop 1 2 3

    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)

    Triple Population and AND Gate

    September 23

    Plasmid Isolation

    • TT-pLsrA-LuxI->>araC
    • TT-pTetR-TetR
    • CP-LsrK->>LuxR
    • TT-pLuxR-LacI

    Digestions and Gel Extraction

    • TT-pLsrA-LuxI->>araC
    • TT-pTetR-TetR
    • CP-LsrK->>LuxR
    • TT-pLuxR-LacI

    Retrieved from "http://2012.igem.org/Team:UT_Dallas/notebook_data"