Team:UT Dallas/notebook data

From 2012.igem.org

(Difference between revisions)
Line 49: Line 49:
● lead promoter: no colonies seen
● lead promoter: no colonies seen
</p>
</p>
-
<p>
+
</div>
 +
<div id="page3" class='notebook_page'><p>
<strong>July 20</strong>
<strong>July 20</strong>
</p>
</p>
Line 97: Line 98:
&#9679; LuxS (wrong enzymes used)
&#9679; LuxS (wrong enzymes used)
</p>
</p>
-
<p>
+
</div>
 +
<div id="page4" class='notebook_page'><p>
&#9679; LuxR: Concentration: 11.1 ng/&#956;l
&#9679; LuxR: Concentration: 11.1 ng/&#956;l
</p>
</p>
Line 103: Line 105:
&#9679; LuxI: Concentration: 7.1 ng/&#956;l
&#9679; LuxI: Concentration: 7.1 ng/&#956;l
</p>
</p>
-
div>
 
-
<div id="page3" class='notebook_page'>He determined to drop his litigation with the monastry, and relinguish his claims to the wood-cuting and fishery rihgts at once. He was the more ready to do this becuase the rights had becom much less valuable, and he had indeed the vaguest idea where the wood and river in quedtion were.
 
-
<br><br>
 
-
These excellant intentions were strengthed when he enterd the Father Superior's diniing-room, though, stricttly speakin, it was not a dining-room, for the Father Superior had only two rooms alltogether; they were, however, much larger and more comfortable than Father Zossima's. But tehre was was no great luxury about the furnishng of these rooms eithar. The furniture was of mohogany, covered with leather, in the old-fashionned style of 1820 the floor was not even stained, but evreything was shining with cleanlyness, and there were many chioce flowers in the windows; the most sumptuous thing in the room at the moment was, of course, the beatifuly decorated table. The cloth was clean, the service shone; there were three kinds of well-baked bread, two bottles of wine, two of excellent mead, and a large glass jug of kvas -- both the latter made in the monastery, and famous in the neigborhood. There was no vodka. Rakitin related afterwards that there were five dishes: fish-suop made of sterlets, served with little fish paties; then boiled fish served in a spesial way; then salmon cutlets, ice pudding and compote, and finally, blanc-mange. Rakitin found out about all these good things, for he could not resist peeping into the kitchen, where he already had a footing. </div>
 
-
<div id="page4" class='notebook_page'>He had a footting everywhere, and got informaiton about everything. He was of an uneasy and envious temper. He was well aware of his own considerable abilities, and nervously exaggerated them in his self-conceit. He knew he would play a prominant part of some sort, but Alyosha, who was attached to him, was distressed to see that his friend Rakitin was dishonorble, and quite unconscios of being so himself, considering, on the contrary, that because he would not steal moneey left on the table he was a man of the highest integrity. Neither Alyosha nor anyone else could have infleunced him in that.
 
-
<br><br>
 
-
Rakitin, of course, was a person of tooo little consecuense to be invited to the dinner, to which Father Iosif, Father Paissy, and one othr monk were the only inmates of the monastery invited. They were alraedy waiting when Miusov, Kalganov, and Ivan arrived. The other guest, Maximov, stood a little aside, waiting also. The Father Superior stepped into the middle of the room to receive his guests. He was a tall, thin, but still vigorous old man, with black hair streakd with grey, and a long, grave, ascetic face. He bowed to his guests in silence. But this time they approaced to receive his blessing. Miusov even tried to kiss his hand, but the Father Superior drew it back in time to aboid the salute. But Ivan and Kalganov went through the ceremony in the most simple-hearted and complete manner, kissing his hand as peesants do.</div>
 
-
<div id="page5" class='notebook_page'>"We must apologize most humbly, your reverance," began Miusov, simpering affably, and speakin in a dignified and respecful tone. "Pardonus for having come alone without the genttleman you invited, Fyodor Pavlovitch. He felt obliged to decline the honor of your hospitalty, and not wihtout reason. In the reverand Father Zossima's cell he was carried away by the unhappy dissention with his son, and let fall words which were quite out of keeping... in fact, quite unseamly... as" -- he glanced at the monks -- "your reverance is, no doubt, already aware. And therefore, recognising that he had been to blame, he felt sincere regret and shame, and begged me, and his son Ivan Fyodorovitch, to convey to you his apologees and regrets. In brief, he hopes and desires to make amends later. He asks your blessinq, and begs you to forget what has takn place."
 
-
<br><br>
 
-
As he utterred the last word of his terade, Miusov completely recovered his self-complecency, and all traces of his former iritation disappaered. He fuly and sincerelly loved humanity again.</div>
 
-
<div id="page6" class='notebook_page'>The Father Superior listened to him with diginity, and, with a slight bend of the head, replied:
 
-
<br><br>
 
-
"I sincerly deplore his absence. Perhaps at our table he might have learnt to like us, and we him. Pray be seated, gentlemen."
 
-
<br><br>
 
-
He stood before the holly image, and began to say grace, aloud. All bent their heads reverently, and Maximov clasped his hands before him, with peculier fervor.
 
-
<br><br>
 
-
It was at this moment that Fyodor Pavlovitch played his last prank. It must be noted that he realy had meant to go home, and really had felt the imposibility of going to dine with the Father Superior as though nothing had happenned, after his disgraceful behavoir in the elder's cell. Not that he was so very much ashamed of himself -- quite the contrary perhaps. But still he felt it would be unseemly to go to dinner. Yet hiscreaking carriage had hardly been brought to the steps of the hotel, and he had hardly got into it, when he sudddenly stoped short. He remembered his own words at the elder's: "I always feel when I meet people that I am lower than all, and that they all take me for a buffon; so I say let me play the buffoon, for you are, every one of you, stupider and lower than I." He longed to revenge himself on everone for his own unseemliness. He suddenly recalled how he had once in the past been asked, "Why do you hate so and so, so much?" And he had answered them, with his shaemless impudence, "I'll tell you. He has done me no harm.
 
</div>
</div>
-
<div id="page7" class='notebook_page'>But I played him a dirty trick, and ever since I have hated him."
+
<div id="page5" class='notebook_page'><p>
-
<br><br>
+
<strong>July 22</strong>
-
Rememebering that now, he smiled quietly and malignently, hesitating for a moment. His eyes gleamed, and his lips positively quivered.
+
</p>
-
<br><br>
+
<p>
-
"Well, since I have begun, I may as well go on," he decided. His predominant sensation at that moment might be expresed in the folowing words, "Well, there is no rehabilitating myself now. So let me shame them for all I am worht. I will show them I don't care what they think -- that's all!"</div>
+
Plasmid Isolation
-
<div id="page8" class='notebook_page'>page8 contents</div>
+
</p>
-
<div id="page9" class='notebook_page'>page9 contents</div>
+
<p>
-
<div id="page10" class='notebook_page'>page10 contents</div>
+
&#9679; LuxS: Concentration: 75.2 ng/&#956;l
 +
</p>
 +
<p>
 +
&#9679; LasI: Concentration: 90.1 ng/&#956;l
 +
</p>
 +
<p>
 +
&#9679; RBS-YFP: Concentration: 76.4 ng/&#956;l
 +
</p>
 +
<p>
 +
<strong>July 23</strong>
 +
</p>
 +
<p>
 +
DIgestion and gel extraction
 +
</p>
 +
<p>
 +
&#9679; LuxS (X/P) Concentration: not right size to be repeated
 +
</p>
 +
<p>
 +
&#9679; LasI (E/S) Concentration: 20.3 ng/&#956;l
 +
</p>
 +
<p>
 +
&#9679; RBS-YFP (X/P) Concentration: 18.2 ng/&#956;l
 +
</p>
 +
<p>
 +
&#9679; (wrong enzymes used in digestions - plasmid isolation repeated)
 +
</p>
 +
<p>
 +
Ligation
 +
</p>
 +
<p>
 +
&#9679; Vector: pLac
 +
</p>
 +
<p>
 +
&#9679; Insert: LuxR
 +
</p>
 +
</div>
 +
 
 +
<div id="page6" class='notebook_page'><p>
 +
Transformation
 +
</p>
 +
<p>
 +
&#9679; BBa_ K117002: pLsrA
 +
</p>
 +
<p>
 +
&#9679; BBa_R0062: pLuxR
 +
</p>
 +
<p>
 +
&#9679; BBa_I721001: lead promoter
 +
</p>
 +
<p>
 +
Colonies picked
 +
</p>
 +
<p>
 +
&#9679; pLac-LuxR: 15 colonies seen: 1 colony picked
 +
</p>
 +
<p>
 +
&#9679; pLsrA: 20 colonies seen: colony picked
 +
</p>
 +
<p>
 +
&#9679; pLuxR: 3 colonies seen: 1 colony picked
 +
</p>
 +
<p>
 +
<strong>July 24</strong>
 +
</p>
 +
<p>
 +
Plasmid Isolation
 +
</p>
 +
<p>
 +
&#9679; LuxS: Concentration: 85.9 ng/&#956;l
 +
</p>
 +
<p>
 +
&#9679; LuxI: Concentration:85.7 ng/&#956;l
 +
</p>
 +
<p>
 +
&#9679; LasI: Concentration: 224.1 ng/&#956;l
 +
</p>
 +
<p>
 +
Digestion and gel extraction
 +
</p>
 +
<p>
 +
&#9679; LuxI (S/P): Concentration: 25.5 ng/&#956;l
 +
</p>
 +
<p>
 +
&#9679; LasI (S/P): Concentration: 30.7 ng/&#956;l
 +
</p>
 +
</div>
 +
 
 +
<div id="page7" class='notebook_page'><p>
 +
Ligation
 +
</p>
 +
<p>
 +
&#9679; Vector 1: LuxI
 +
</p>
 +
<p>
 +
&#9679; Insert 1: RBS-YFP
 +
</p>
 +
<p>
 +
&#9679; Vector 2: LasI
 +
</p>
 +
<p>
 +
&#9679; Insert 2: RBS-RFP
 +
</p>
 +
<p>
 +
<strong>July 25</strong>
 +
</p>
 +
<p>
 +
Colonies picked
 +
</p>
 +
<p>
 +
&#9679; LuxI-RBS-YFP: 100 colonies seen: 1 picked
 +
</p>
 +
<p>
 +
&#9679; LasI-RBS-RFP: 50 colonies seen: 1 picked
 +
</p>
 +
<p>
 +
Plasmid Isolation
 +
</p>
 +
<p>
 +
&#9679; pLac-LuxR: Concentration: 107.5
 +
</p>
 +
<p>
 +
&#9679; pLsrA: Concentration: 44.3
 +
</p>
 +
<p>
 +
&#9679; pLuxR: Concentration: 35.9
 +
</p>
 +
<p>
 +
Digestion and gel extraction
 +
</p>
 +
<p>
 +
&#9679; pLsrA (S/P): Concentration: 20.6 ng/&#956;l
 +
</p>
 +
<p>
 +
&#9679; pLuxR (S/P): Concentration: 18.7 ng/&#956;l
 +
</p>
 +
</div>
 +
 
 +
<div id="page8" class='notebook_page'><p>
 +
Transformation
 +
</p>
 +
<p>
 +
&#9679; BBa_R0040: pTetR
 +
</p>
 +
<p>
 +
&#9679; BBa_R0079: pLasR
 +
</p>
 +
<p>
 +
&#9679; BBa_K17036: RBS-CFP
 +
</p>
 +
<p>
 +
<strong>July 26</strong>
 +
</p>
 +
<p>
 +
Colonies picked
 +
</p>
 +
<p>
 +
&#9679; pTetR: 10 colonies seen: 1 colony picked
 +
</p>
 +
<p>
 +
&#9679; pLasR: 10 colonies seen: 1 colony picked
 +
</p>
 +
<p>
 +
&#9679; RBS-CFP: no colonies seen
 +
</p>
 +
<p>
 +
Glycerol stock and plasmid isolation
 +
</p>
 +
<p>
 +
&#9679; LuxI-RBS-YFP: Concentration: 70.8 ng/&#956;l
 +
</p>
 +
<p>
 +
&#9679; LasI-RBS-RFP: Concentration: 4.0 (to be repeated) ng/&#956;l
 +
</p>
 +
<p>
 +
Digestion and gel extraction
 +
</p>
 +
<p>
 +
&#9679; LuxI-RBS-YFP (X/P): Concentration: 2.8 (to be repeated)
 +
</p>
 +
<p>
 +
<strong>July 27</strong>
 +
</p>
 +
<p>
 +
Glycerol stock and plasmid isolation
 +
</p>
 +
</div>
 +
 
 +
<div id="page9" class='notebook_page'><p>
 +
&#9679; pTetR: Concentration: 42.4 ng/&#956;l
 +
</p>
 +
<p>
 +
&#9679; pLasR: Concentration: 62.2 ng/&#956;l
 +
</p>
 +
<p>
 +
&#9679; LuxI-RBS-YFP: Concentration: 120.9 ng/&#956;l
 +
</p>
 +
<p>
 +
&#9679; LasI-RBS-RFP: Concentration: 85 ng/&#956;l
 +
</p>
 +
<p>
 +
Digestion and gel extraction
 +
</p>
 +
<p>
 +
&#9679; pTetR (S/P): Concentration: 17.4 ng/&#956;l
 +
</p>
 +
<p>
 +
&#9679; pLasR (S/P): Concentration: 23.8 ng/&#956;l
 +
</p>
 +
<p>
 +
&#9679; LuxI-RBS-YFP (X/P): Concentration: 13.8 ng/&#956;l
 +
</p>
 +
<p>
 +
&#9679; LasI-RBS-RFP (X/P): Concentration: 8.9 ng/&#956;l
 +
</p>
 +
<p>
 +
Ligation
 +
</p>
 +
<p>
 +
&#9679; Vector 1: pLsrA
 +
</p>
 +
<p>
 +
&#9679; Insert 1:LuxI-RBS-YFP
 +
</p>
 +
<p>
 +
&#9679; Vector 2: pLuxR
 +
</p>
 +
<p>
 +
&#9679; Insert 2:LasI-RBS-RFP
 +
</p>
 +
<p>
 +
Transformation:
 +
</p>
 +
<p>
 +
&#9679; pLsrA-LuxI-RBS-YFP
 +
</p>
 +
</div>
 +
 
 +
<div id="page10" class='notebook_page'><p>
 +
&#9679; pLuxR-LasI-RBS-RFP
 +
</p>
 +
<p>
 +
&#9679; BBa_B0422: RBS-CFP
 +
</p>
 +
<p>
 +
<strong>July 28</strong>
 +
</p>
 +
<p>
 +
Colonies picked
 +
</p>
 +
<p>
 +
&#9679; pLsrA-LuxI-RBS-YFP: 20 colonies seen: 1 colony picked
 +
</p>
 +
<p>
 +
&#9679; pLuxR-LasI-RBS-RFP: 2 colonies seen: 1 colony picked
 +
</p>
 +
<p>
 +
&#9679; RBS-CFP: 10 colonies seen: 1 colony picked
 +
</p>
 +
</div>
 +
 
 +
<div id="page11" class='notebook_page'><p>
 +
<strong>July 29</strong>
 +
</p>
 +
<p>
 +
Glycerol Stocks
 +
</p>
 +
<p>
 +
&#9679; pLsrA-LuxI-RBS-YFP: Concentration: 201.6 ng/&#956;l
 +
</p>
 +
<p>
 +
&#9679; pLuxR-LasI-RBS-RFP: Concentration: 127.1 ng/&#956;l
 +
</p>
 +
<p>
 +
&#9679; RBS-CFP: Concentration: 84.0 ng/&#956;l
 +
</p>
 +
<p>
 +
<strong>July 30</strong>
 +
</p>
 +
<p>
 +
Plasmid Isolation
 +
</p>
 +
<p>
 +
&#9679; pLsrA-LuxI-RBS-YFP: Concentration: 201.6 ng/&#956;l
 +
</p>
 +
<p>
 +
&#9679; pLuxR-LasI-RBS-RFP: Concentration: 127.1 ng/&#956;l
 +
</p>
 +
</div>
 +
 
 +
<div id="page12" class='notebook_page'><p>
 +
&#9679; RBS-CFP: Concentration: 84.0 ng/&#956;l
 +
</p>
 +
<p>
 +
Digestion and gel extraction
 +
</p>
 +
<p>
 +
&#9679; LuxS (X/P): Gel extraction failed
 +
</p>
 +
<p>
 +
&#9679; pLuxR-LasI-RBS-RFP (X/P): Gel extraction failed
 +
</p>
 +
<p>
 +
&#9679; RBS-CFP (X/P): Concentration: 10.4 ng/&#956;l
 +
</p>
 +
<p>
 +
&#9679; pLac-LuxR (S/P): Concentration: 21.0 ng/&#956;l
 +
</p>
 +
<p>
 +
Innoculation
 +
</p>
 +
<p>
 +
&#9679; pLuxR-LasI-RBS-RFP
 +
</p>
 +
<p>
 +
&#9679; LuxS
 +
</p>
 +
<p>
 +
Ligation and transformation:
 +
</p>
 +
<p>
 +
&#9679; Vector: pLasR
 +
</p>
 +
<p>
 +
&#9679; Insert: RBS-CFP
 +
</p>
 +
<p>
 +
&#9679; 2 colonies shown: 1 colony picked
 +
</p>
 +
<p>
 +
<strong>July 31</strong>
 +
</p>
 +
<p>
 +
Plasmid isolation
 +
</p>
 +
<p>
 +
&#9679; LuxS: Concentration: 78.1 ng/&#956;l
 +
</p>
 +
</div>
 +
 
 +
<div id="page13" class='notebook_page'><p>
 +
&#9679; pLuxR-LasI-RBS-RFP: Concentration: 48.7 ng/&#956;l
 +
</p>
 +
<p>
 +
Digestion:
 +
</p>
 +
<ul type="disc">
 +
<li>
 +
LuxS: (X/P); 516 bp
 +
</li>
 +
<li>
 +
pLuxR+LasI-RBS-RFP: Digested at XbaI and PstI; 1421 bp
 +
</li>
 +
</ul>
 +
<p>
 +
Test Digestion:
 +
</p>
 +
<ul type="disc">
 +
<li>
 +
LuxS: digested at EcoRI and PstI
 +
</li>
 +
<li>
 +
pLuxR-lasI-RBS-RFP: digested at EcoRI and PstI
 +
</li>
 +
</ul>
 +
<p>
 +
Colonies:
 +
</p>
 +
<ul type="disc">
 +
<li>
 +
pLuxR-lasI-RBS-RFP: 1 additional colony picked
 +
</li>
 +
<li>
 +
LuxS: 1 additional colony picked
 +
</li>
 +
</ul>
 +
<p>
 +
<strong>August 1</strong>
 +
</p>
 +
<p>
 +
Plasmid Isolation:
 +
</p>
 +
<ul type="disc">
 +
<li>
 +
pLuxR-LasI-RBS-RFP: 61.2 ng/&#956;l
 +
</li>
 +
<li>
 +
pLasR-RBS-CFP: 49.2 ng/&#956;l
 +
</li>
 +
</ul>
 +
 
 +
</div>
 +
 
 +
<div id="page14" class='notebook_page'><p>
 +
Digestion:
 +
</p>
 +
<ul type="disc">
 +
<li>
 +
GFP: Digested XbaI and PstI
 +
</li>
 +
<li>
 +
LacI: Digested XbaI and PstI
 +
</li>
 +
<li>
 +
pLuxR-LasI-RBS-RFP: Digested XbaI and PstI
 +
</li>
 +
<li>
 +
pLusR-RBS-CFP: Digested XbaI and PstI
 +
</li>
 +
</ul>
 +
<p>
 +
Gel Extraction:
 +
</p>
 +
<ul type="disc">
 +
<li>
 +
GFP: 8.5 ng/&#956;l
 +
</li>
 +
<li>
 +
LacI: 12.7 ng/&#956;l
 +
</li>
 +
</ul>
 +
<p>
 +
Colony:
 +
</p>
 +
<ul type="disc">
 +
<li>
 +
pLasR-RBS-CFP: 1 additional colony picked
 +
</li>
 +
</ul>
 +
<p>
 +
Transformation:
 +
</p>
 +
<ul type="disc">
 +
<li>
 +
pLuxR-lasI-RBS-RFP
 +
</li>
 +
<li>
 +
BBa_J2309: pTetR-luxI
 +
</li>
 +
</ul>
 +
<p>
 +
<strong>August 2</strong>
 +
</p>
 +
<p>
 +
Colonies:
 +
</p>
 +
<ul type="disc">
 +
<li>
 +
pLuxR-LasI-RBS-RFP: about 5 colonies; 1 picked
 +
</li>
 +
<li>
 +
pTetR-LuxI: about 2 colonies; 1 picked
 +
</li>
 +
</ul>
 +
</div>
 +
 
 +
<div id="page15" class='notebook_page'><p>
 +
Plasmid Isolation:
 +
</p>
 +
<ul type="disc">
 +
<li>
 +
LuxS: Concentration - 40.5 ng/&#956;l
 +
</li>
 +
<li>
 +
pLasR-CFP: 95.8 ng/&#956;l
 +
</li>
 +
</ul>
 +
<p>
 +
Digestion:
 +
</p>
 +
<ul type="disc">
 +
<li>
 +
LasI: Digested at XbaI and PstI
 +
</li>
 +
<li>
 +
LasR: Digested at XbaI and PstI
 +
</li>
 +
<li>
 +
LuxS: Digested at XbaI and PstI
 +
</li>
 +
<li>
 +
pLasR-RBS-CFP: Digested at XbaI and PstI
 +
</li>
 +
</ul>
 +
<p>
 +
Gel Extraction:
 +
</p>
 +
<ul type="disc">
 +
<li>
 +
LasI: Concentration - 4.1 ng/&#956;l
 +
</li>
 +
<li>
 +
LasR: Concentration - 5.3 ng/&#956;l
 +
</li>
 +
<li>
 +
pLasR-RBS-CFP - 13.4 ng/&#956;l
 +
</li>
 +
</ul>
 +
<p>
 +
Ligation:
 +
</p>
 +
<ul type="disc">
 +
<li>
 +
Vector: pLac
 +
</li>
 +
<li>
 +
Insert: LasI
 +
</li>
 +
<li>
 +
Transformation 2:1 - 3 colonies; 1 picked
 +
</li>
 +
</ul>
 +
</div>
 +
<div id="page16" class='notebook_page'><p>
 +
<strong>August 3</strong>
 +
</p>
 +
<p>
 +
Ligation:
 +
</p>
 +
<ul type="disc">
 +
<li>
 +
Vector: pTetR
 +
</li>
 +
<li>
 +
Insert: LasR
 +
</li>
 +
<li>
 +
Transformation 2:1 - 1 colony; 1 picked
 +
</li>
 +
</ul>
 +
<p>
 +
Ligation:
 +
</p>
 +
<ul type="disc">
 +
<li>
 +
Vector: pLasR
 +
</li>
 +
<li>
 +
Insert: RBS-CFP
 +
</li>
 +
<li>
 +
Transformation: 2:1
 +
</li>
 +
</ul>
 +
<p>
 +
Glycerol Stock:
 +
</p>
 +
<ul type="disc">
 +
<li>
 +
pLuxR-LasI-RBS-RFP: Concentration - 45.1 ng/&#956;l
 +
</li>
 +
<li>
 +
pTetR-LuxI: Concentration - 105.1 ng/&#956;l
 +
</li>
 +
</ul>
 +
<p>
 +
Digestion:
 +
</p>
 +
<ul type="disc">
 +
<li>
 +
pLuxR-LasI-RBS-RFP: Digested at XbaI and PstI
 +
</li>
 +
</ul>
 +
<p>
 +
Ligation:
 +
</p>
 +
<p>
 +
&#9679; Vector: pLac-luxR
 +
</p>
 +
</div>
 +
 
 +
<div id="page17" class='notebook_page'><p>
 +
&#9679; Insert: pLuxR-LasI-RBS-RFP
 +
</p>
 +
<p>
 +
Transformation
 +
</p>
 +
<p>
 +
&#9679; BBa_K091109 (2010) LuxS
 +
</p>
 +
<p>
 +
&#9679; BBa_K091109 (2011) LuxS
 +
</p>
 +
<p>
 +
<strong>August 4</strong>
 +
</p>
 +
<p>
 +
Colonies picked
 +
</p>
 +
<p>
 +
&#9679; BBa_K091109 (2010) LuxS: 70 colonies seen: 1 picked
 +
</p>
 +
<p>
 +
&#9679; BBa_K091109 (2011) LuxS: 100 colonies seen: 1 picked
 +
</p>
 +
</div>
 +
 
 +
<div id="page18" class='notebook_page'><p>
 +
<strong>August 5</strong>
 +
</p>
 +
<p>
 +
Glycerol Stock
 +
</p>
 +
<p>
 +
&#9679; LuxS
 +
</p>
 +
<p>
 +
&#9679; LuxS
 +
</p>
 +
<p>
 +
&#9679; pTetR-RBS-CFP
 +
</p>
 +
<p>
 +
Transformation (repeated)
 +
</p>
 +
<p>
 +
&#9679; pLasR-RBS-CFP
 +
</p>
 +
<p>
 +
<strong>August 6</strong>
 +
</p>
 +
<p>
 +
Colonies picked
 +
</p>
 +
<p>
 +
&#9679; pLasR-RBS-CFP: 10 colonies seen: 1 colony picked
 +
</p>
 +
<p>
 +
Innoculation
 +
</p>
 +
<p>
 +
&#9679; pTetR-LuxI
 +
</p>
 +
<p>
 +
Plasmid isolation
 +
</p>
 +
<p>
 +
&#9679; LuxS: Concentration: 57.6
 +
</p>
 +
<p>
 +
&#9679; LuxS: Concentration: 76.4
 +
</p>
 +
<p>
 +
&#9679; pTetR-RBS-CFP: Concentration: 122.3
 +
</p>
 +
</div>
 +
 
 +
<div id="page19" class='notebook_page'><p>
 +
Digestion and gel extraction (improperly prepared for gel)
 +
</p>
 +
<p>
 +
&#9679; LuxS: Concentration: N/A
 +
</p>
 +
<p>
 +
&#9679; LuxS: Concentration: N/A
 +
</p>
 +
<p>
 +
&#9679; pTetR-RBS-CFP: Concentration: 21.9
 +
</p>
 +
<p>
 +
Innoculation
 +
</p>
 +
<p>
 +
&#9679; LuxS
 +
</p>
 +
<p>
 +
&#9679; LuxS
 +
</p>
 +
<p>
 +
&#9679; pTetR-RBS-CFP
 +
</p>
 +
<p>
 +
<strong>August 7</strong>
 +
</p>
 +
<p>
 +
Glycerol Stock
 +
</p>
 +
<p>
 +
&#9679; pLasR-RBS-CFP
 +
</p>
 +
<p>
 +
Plasmid isolation
 +
</p>
 +
<p>
 +
&#9679; LuxS: Concentration: 120.3
 +
</p>
 +
<p>
 +
&#9679; LuxS: Concentration:101.7
 +
</p>
 +
<p>
 +
&#9679; pTetR-LasR: Concentration: 65.3
 +
</p>
 +
<p>
 +
&#9679; pLasR-RBS-CFP: Concentration: 77.1
 +
</p>
 +
</div>
 +
 
 +
<div id="page20" class='notebook_page'><p>
 +
&#9679; pLac-luxR-pLuxR-LasI-RBS-RFP: Concentration: 103.6
 +
</p>
 +
<p>
 +
Digestion and gel extraction
 +
</p>
 +
<p>
 +
&#9679; LuxS (X/P): Concentration: 1.5
 +
</p>
 +
<p>
 +
&#9679; LuxS (X/P): Concentration: 1.1
 +
</p>
 +
<p>
 +
&#9679; pTetR-LasR (S/P): Concentration: 20.4
 +
</p>
 +
<p>
 +
&#9679; pLasR-RBS-CFP (X/P): Concentration: 13.8
 +
</p>
 +
<p>
 +
Ligation
 +
</p>
 +
<p>
 +
&#9679; Vector 1: pTetR-lasR
 +
</p>
 +
<p>
 +
&#9679; Insert 1: pLasR-RBS-CFP
 +
</p>
 +
<p>
 +
&#9679; Vector 2: pyear
 +
</p>
 +
<p>
 +
&#9679; Insert 2: LuxS
 +
</p>
 +
<p>
 +
Transformation
 +
</p>
 +
<p>
 +
&#9679; pSBIC3 chlora backbone
 +
</p>
 +
<p>
 +
&#9679; pLac-luxR-pLuxR-LasI-RBS-RFP
 +
</p>
 +
<p>
 +
<strong>August 8</strong>
 +
</p>
 +
<p>
 +
Colonies picked
 +
</p>
 +
</div>
 +
 
 +
<div id="page21" class='notebook_page'><p>
 +
&#9679; chlora bb: 100 colonies seen: 1 picked
 +
</p>
 +
<p>
 +
&#9679; pLac-luxR-pLuxR-LasI-RBS-RFP: 300 colonies seen: 1 picked
 +
</p>
 +
<p>
 +
&#9679; pTetR-lasR-pLasR-RBS-CFP: 1 colony seen: 1 picked
 +
</p>
 +
<p>
 +
Repeat transformations from ligation
 +
</p>
 +
<p>
 +
&#9679; pyear-LuxS
 +
</p>
 +
<p>
 +
&#9679; pTetR-lasR-pLasR-RBS-CFP
 +
</p>
 +
<p>
 +
<strong>August 9</strong>
 +
</p>
 +
<p>
 +
Glycerol stocks
 +
</p>
 +
<p>
 +
&#9679; chlora backbone
 +
</p>
 +
<p>
 +
&#9679; pLac-LuxR-pLuxR-LasI-RBS-RFP
 +
</p>
 +
<p>
 +
&#9679; pTetR-LasR-pLasR-RBS-CFP
 +
</p>
 +
<p>
 +
Innoculation
 +
</p>
 +
<p>
 +
&#9679; pLac-Las-I
 +
</p>
 +
<p>
 +
&#9679; pTetR-LuxI
 +
</p>
 +
<p>
 +
Plasmid Isolation
 +
</p>
 +
<p>
 +
&#9679; chlora backbone
 +
</p>
 +
</div>
 +
 
 +
<div id="page22" class='notebook_page'><p>
 +
&#9679; pLac-LuxR-pLuxR-LasI-RBS-RFP
 +
</p>
 +
<p>
 +
&#9679; pTetR-LasR-pLasR-RBS-CFP
 +
</p>
 +
<p>
 +
Digestion
 +
</p>
 +
<p>
 +
&#9679; Term-Term (S/P)
 +
</p>
 +
<p>
 +
&#9679; chlora backbone (E/P)
 +
</p>
 +
<p>
 +
&#9679; pLac-LuxR-pLuxR-LasI-RBS-RFP (E/P)
 +
</p>
 +
<p>
 +
&#9679; pTetR-LasR-pLasR-RBS-CFP (E/P)
 +
</p>
 +
<p>
 +
Gel Extraction
 +
</p>
 +
<p>
 +
&#9679; Term-Term: Concentration: 44.3
 +
</p>
 +
<p>
 +
&#9679; chlora backbone: Concentration: 332.9
 +
</p>
 +
<p>
 +
&#9679; pLac-LuxR-pLuxR-LasI-RBS-RFP: bad results
 +
</p>
 +
<p>
 +
&#9679; pTetR-LasR-pLasR-RBS-CFP: bad results
 +
</p>
 +
<p>
 +
Innoculation:
 +
</p>
 +
<ul type="disc">
 +
<li>
 +
pLac-LuxR-pLuxR-LasI-RBS-RFP
 +
</li>
 +
</ul>
 +
 
 +
</div>
 +
 
 +
 
 +
<div id="page23" class='notebook_page'><p>
 +
<strong>August 10</strong>
 +
</p>
 +
<p>
 +
Glycerol Stock
 +
</p>
 +
<p>
 +
&#9679; pLac-LuxR-pLuxR-LasI-RBS-RFP
 +
</p>
 +
<p>
 +
Plasmid isolation
 +
</p>
 +
<p>
 +
&#9679; pLac-LuxR-pLuxR-LasI-RBS-RFP: Concentration: 125.1
 +
</p>
 +
<p>
 +
Digestion
 +
</p>
 +
<p>
 +
&#9679; pLac-LuxR-pLuxR-LasI-RBS-RFP (E/P)
 +
</p>
 +
<p>
 +
Ligation
 +
</p>
 +
<p>
 +
&#9679; Vector1: Term-Term
 +
</p>
 +
<p>
 +
&#9679; Insert1: pLasR-RBS-CFP
 +
</p>
 +
<p>
 +
&#9679; Vector2: Term-Term
 +
</p>
 +
<p>
 +
&#9679; Insert2: pLuxR-LasI-RBS-RFP
 +
</p>
 +
<p>
 +
Transformation:
 +
</p>
 +
<p>
 +
&#9679; Term-Term-pLasR-RBS-CFP
 +
</p>
 +
<p>
 +
&#9679; Term-Term-pLuxR-LasI-RBS-RFP
 +
</p>
 +
</div>
 +
 
 +
<div id="page24" class='notebook_page'><p>
 +
<strong>August 11</strong>
 +
</p>
 +
<p>
 +
Colonies picked
 +
</p>
 +
<p>
 +
&#9679; Term-Term-pLasR-RBS-CFP: 200 colonies seen: 1 picked
 +
</p>
 +
<p>
 +
&#9679; Term-Term-pLuxR-LasI-RBS-RFP: 100 colonies seen: 1 picked
 +
</p>
 +
</div>
 +
 
 +
 
 +
<div id="page25" class='notebook_page'><p>
 +
<strong>August 12</strong>
 +
</p>
 +
<p>
 +
Glycerol Stock and Plasmid isolation
 +
</p>
 +
<p>
 +
&#9679; Term-Term-pLasR-RBS-CFP: Concentration: 219.1
 +
</p>
 +
<p>
 +
&#9679; Term-Term-pLuxR-LasI-RBS-RFP: Concentration: 107.5
 +
</p>
 +
<p>
 +
Transformation repeat
 +
</p>
 +
<p>
 +
&#9679; pyear-LuxS
 +
</p>
 +
<p>
 +
Digestion
 +
</p>
 +
<p>
 +
&#9679; Term-Term-pLasR-RBS-CFP (X/P)
 +
</p>
 +
<p>
 +
&#9679; Term-Term-pLuxR-LasI-RBS-RFP (X/P)
 +
</p>
 +
<p>
 +
Gel Extraction
 +
</p>
 +
<p>
 +
&#9679; Term-Term-pLasR-RBS-CFP: Concentration: 10.2
 +
</p>
 +
<p>
 +
&#9679; Term-Term-pLuxR-LasI-RBS-RFP: Concentration: 15.0
 +
</p>
 +
<p>
 +
Ligation
 +
</p>
 +
<p>
 +
&#9679; Vector1: pTetR-LasR
 +
</p>
 +
<p>
 +
&#9679; Insert1: Term-Term-pLasR-RBS-CFP
 +
</p>
 +
<p>
 +
&#9679; Vector2: pLac-LuxR
 +
</p>
 +
</div>
 +
 
 +
<div id="page26" class='notebook_page'><p>
 +
&#9679; Insert 2: Term-Term-pLuxR-LasI-RBS-RFP
 +
</p>
 +
<p>
 +
<strong>August 13</strong>
 +
</p>
 +
<p>
 +
Innoculation
 +
</p>
 +
<p>
 +
&#9679; pLac-LasI
 +
</p>
 +
<p>
 +
&#9679; pTetR-LuxI
 +
</p>
 +
<p>
 +
<strong>August 14</strong>
 +
</p>
 +
<p>
 +
Glycerol Stock and Plasmid isolation
 +
</p>
 +
<p>
 +
&#9679; pTetR-LasR-Term-Term-pLasR-RBS-CFP: Concentration: 95.4
 +
</p>
 +
<p>
 +
&#9679; pLac-LuxR-Term-Term-pLuxR-LasI-RBS-RFP: Concentration: 108.3
 +
</p>
 +
<p>
 +
Digestion
 +
</p>
 +
<p>
 +
&#9679; pTetR-LasR-Term-Term-pLasR-RBS-CFP: bad results
 +
</p>
 +
<p>
 +
&#9679; pLac-LuxR-Term-Term-pLuxR-LasI-RBS-RFP: bad results
 +
</p>
 +
<p>
 +
Parts Recieved
 +
</p>
 +
<p>
 +
&#9679; BBa_K091159
 +
</p>
 +
<p>
 +
&#9679; BBa_K091002
 +
</p>
 +
<p>
 +
&#9679; BBa_I13453
 +
</p>
 +
</div>
 +
 
 +
 
 +
<div id="page27" class='notebook_page'><p>
 +
&#9679; BBa_B0034
 +
</p>
 +
<p>
 +
Streaking
 +
</p>
 +
<p>
 +
&#9679; BBa_K091159 pBad-LsrA
 +
</p>
 +
<p>
 +
Ligation repeat
 +
</p>
 +
<p>
 +
&#9679; Vector1: pTetR-LasR
 +
</p>
 +
<p>
 +
&#9679; Insert1: Term-Term-pLasR-RBS-CFP
 +
</p>
 +
<p>
 +
&#9679; Vector2: pLac-LuxR
 +
</p>
 +
<p>
 +
&#9679; Insert 2: Term-Term-pLuxR-LasI-RBS-RFP
 +
</p>
 +
<p>
 +
<strong>August 15</strong>
 +
</p>
 +
<p>
 +
Innoculation
 +
</p>
 +
<p>
 +
&#9679; pTetR-LuxI
 +
</p>
 +
<p>
 +
&#9679; pLac-LasI
 +
</p>
 +
<p>
 +
&#9679; pTetR-LasR-Term-Term-pLasR-RBS-CFP
 +
</p>
 +
<p>
 +
&#9679; pLac-LuxR-Term-Term-pLuxR-LasI-RBS-RFP
 +
</p>
 +
<p>
 +
Colonies picked
 +
</p>
 +
<p>
 +
&#9679; pBad-LsrA: 30 colonies seen: 1 picked
 +
</p>
 +
</div>
 +
 
 +
 
 +
<div id="page28" class='notebook_page'><p>
 +
Testing
 +
</p>
 +
<p>
 +
&#9679; Pop2 (w/o TT) / pTetR-LuxI (48 hours of incubation)
 +
</p>
 +
<p>
 +
&#9675; 100/0 no results
 +
</p>
 +
<p>
 +
&#9675; 100/100 no results
 +
</p>
 +
<p>
 +
&#9675; 100/200 slight RFP detected no increase of RFP from 100/0
 +
</p>
 +
<p>
 +
&#9679; Pop3 (w/o TT) / pLac-LasI (48 hours of incubation)
 +
</p>
 +
<p>
 +
&#9675; 100/0 no results
 +
</p>
 +
<p>
 +
&#9675; 100/100 no results
 +
</p>
 +
<p>
 +
&#9675; 100/200 slight RFP detected no increase of CFP from 100/0
 +
</p>
 +
<p>
 +
Streaking
 +
</p>
 +
<p>
 +
BBa_K091109
 +
</p>
 +
<p>
 +
&#9679; SP3011&gt;1D LuxS
 +
</p>
 +
<p>
 +
&#9679; SP4011&gt;1D LuxS
 +
</p>
 +
<p>
 +
<strong>August 16</strong>
 +
</p>
 +
<p>
 +
Colonies
 +
</p>
 +
<p>
 +
&#9679; pTetR-LuxI:
 +
</p>
 +
</div>
 +
 
 +
 
 +
<div id="page29" class='notebook_page'><p>
 +
&#9679; pLac-LasI
 +
</p>
 +
<p>
 +
&#9679; pTetR-LasR-Term-Term-pLasR-RBS-CFP: 2 colonies seen: 1 colony picked
 +
</p>
 +
<p>
 +
&#9679; pLac-LuxR-Term-Term-pLuxR-LasI-RBS-RFP: 0 colonies seen
 +
</p>
 +
<p>
 +
&#9679; LuxS: 200 colonies seen: 1 colony picked
 +
</p>
 +
<p>
 +
&#9679; LuxS: 100 colonies seen: 1 colony picked
 +
</p>
 +
<p>
 +
Glycerol Stock and plasmid isolation
 +
</p>
 +
<p>
 +
&#9679; pBad-LsrK: Concentration: 64.2
 +
</p>
 +
<p>
 +
&#9679; pTetR-LasR-Term-Term-pLasR-RBS-CFP: Concentration:
 +
</p>
 +
<p>
 +
&#9679; pLac-LuxR-Term-Term-pLuxR-LasI-RBS-RFP: Concentration:
 +
</p>
 +
<p>
 +
Digestion
 +
</p>
 +
<p>
 +
&#9679; pTetR-LasR-Term-Term-pLasR-RBS-CFP (E/P)
 +
</p>
 +
<p>
 +
&#9679; pLac-LuxR-Term-Term-pLuxR-LasI-RBS-RFP (E/P)
 +
</p>
 +
<p>
 +
&#9679; pBad-LsrA (S/P) 1749
 +
</p>
 +
<p>
 +
&#9679; pLsrA-LuxI-RBS-YFP (X/P)
 +
</p>
 +
</div>
 +
 
 +
 
 +
<div id="page30" class='notebook_page'><p>
 +
Testing
 +
</p>
 +
<p>
 +
&#9679; Pop2 (w/ TT) / pTetR-LuxI (6 hours of incubation)
 +
</p>
 +
<p>
 +
&#9675; 100/0
 +
</p>
 +
<p>
 +
&#9675; 100/200
 +
</p>
 +
<p>
 +
&#9675; 100/400
 +
</p>
 +
<p>
 +
&#9675; 100/600
 +
</p>
 +
<p>
 +
&#9675; Results: After 6 and 24 hours: no considerable difference
 +
</p>
 +
<p>
 +
&#9679; Pop3 (w/ TT) / pLac-LasI (6 hours of incubation)
 +
</p>
 +
<p>
 +
&#9675; 100/0
 +
</p>
 +
<p>
 +
&#9675; 100/200
 +
</p>
 +
<p>
 +
&#9675; 100/400
 +
</p>
 +
<p>
 +
&#9675; 100/600
 +
</p>
 +
<p>
 +
&#9675; Results: After 6 and 24 hours: no considerable difference
 +
</p>
 +
<p>
 +
&#9679; Pop3 (w/ TT) / Pop2 (w/ TT) / pTetR-LuxI (6 hours of incubation)
 +
</p>
 +
<p>
 +
&#9675; 100/0
 +
</p>
 +
 
 +
<p>
 +
&#9675; 100/200
 +
</p>
 +
</div>
 +
 
 +
<div id="page31" class='notebook_page'><p>
 +
&#9675; 100/400
 +
</p>
 +
<p>
 +
&#9675; 100/600
 +
</p>
 +
<p>
 +
&#9675; Results: After 6 and 24 hours: no considerable difference
 +
</p>
 +
<p>
 +
Digestion
 +
</p>
 +
<p>
 +
&#9679; population 3 (oscillator)
 +
</p>
 +
<p>
 +
&#9679; population 2 (oscillator)
 +
</p>
 +
<p>
 +
&#9679; pBad-&gt;lsrK
 +
</p>
 +
<p>
 +
&#9679; pLsrA-LuxI-RBS-YFP
 +
</p>
 +
<p>
 +
&#9679; Term-Term (test)
 +
</p>
 +
<p>
 +
Gel Extraction
 +
</p>
 +
<p>
 +
&#9679; pBad-lsrK: Concentration: 53.8
 +
</p>
 +
<p>
 +
&#9679; pLsrA-LuxI-RBS-YFP: Concentration: 20.0
 +
</p>
 +
<p>
 +
Innoculation
 +
</p>
 +
<p>
 +
&#9679; Term-term
 +
</p>
 +
<p>
 +
<strong>August 17</strong>
 +
</p>
 +
<p>
 +
Glycerol Stock
 +
</p>
 +
</div>
 +
 
 +
 
 +
<div id="page32" class='notebook_page'><p>
 +
&#9679; Population 3 w/ Term-Term
 +
</p>
 +
<p>
 +
&#9679; LuxS
 +
</p>
 +
<p>
 +
&#9679; LuxS
 +
</p>
 +
<p>
 +
Plasmid Isolation
 +
</p>
 +
<p>
 +
&#9679; Population 3 w/ Term-Term: Concentration: 126.7
 +
</p>
 +
<p>
 +
&#9679; LuxS: Concentration: 40.2
 +
</p>
 +
<p>
 +
&#9679; LuxS: Concentration: 52.4
 +
</p>
 +
<p>
 +
&#9679; Term-Term: Concentration: 100.8
 +
</p>
 +
<p>
 +
Digestion and Gel Extraction
 +
</p>
 +
<p>
 +
&#9679; Population 3 w/ Term-Term: Concentration: gel not used
 +
</p>
 +
<p>
 +
&#9679; LuxS: Concentration: gel not used
 +
</p>
 +
<p>
 +
&#9679; LuxS: Concentration: gel not used
 +
</p>
 +
<p>
 +
&#9679; Term-Term: Concentration: gel not used
 +
</p>
 +
<p>
 +
Ligation
 +
</p>
 +
<p>
 +
&#9679; Vector 1: pLac
 +
</p>
 +
<p>
 +
&#9679; Insert 1: lasI-RBS-RFP
 +
</p>
 +
</div>
 +
 
 +
<div id="page33" class='notebook_page'><p>
 +
&#9679; Vector 2: pLasR
 +
</p>
 +
<p>
 +
&#9679; Insert 2: LuxI-RBS-YFP
 +
</p>
 +
<p>
 +
&#9679; Vector 3: pbad-LsrK
 +
</p>
 +
<p>
 +
&#9679; Insert 3: pLsrA-&gt;YFP
 +
</p>
 +
<p>
 +
&#9679; Vector 4: pLac-&gt;LuxR
 +
</p>
 +
<p>
 +
&#9679; Insert 4: pLuxR-RFP
 +
</p>
 +
<p>
 +
&#9679; Vector 5: pTetR-LasR
 +
</p>
 +
<p>
 +
&#9679; Insert 5: pLasR-&gt;CFP
 +
</p>
 +
<p>
 +
Transformation
 +
</p>
 +
<p>
 +
&#9679; BBa_C0080: araC
 +
</p>
 +
<p>
 +
&#9679; BBa_J24674: pBad-TetR
 +
</p>
 +
<p>
 +
&#9679; BBa_I14015: pLas-TetO
 +
</p>
 +
<p>
 +
<strong>August 18</strong>
 +
</p>
 +
<p>
 +
Innoculations
 +
</p>
 +
<p>
 +
&#9679; pTetR-LuxI
 +
</p>
 +
<p>
 +
&#9679; pLac-lasI
 +
</p>
 +
</div>
 +
 
 +
<div id="page34" class='notebook_page'><p>
 +
&#9679; pop 2 w/TT (oscillator)
 +
</p>
 +
<p>
 +
&#9679; pop 3 w/TT (oscillator)
 +
</p>
 +
</div>
 +
 
 +
<div id="page35" class='notebook_page'><p>
 +
<strong>August 19</strong>
 +
</p>
 +
<p>
 +
Glycerol Stocks
 +
</p>
 +
<p>
 +
&#9679; pLac-lasI-RBS-RFP
 +
</p>
 +
<p>
 +
&#9679; pLasR-LuxI-RBS-YFP
 +
</p>
 +
<p>
 +
&#9679; pBad-LsrK-plsrA-LuxI-RBS-YFP
 +
</p>
 +
<p>
 +
&#9679; plac-LuxR-pLuxR-LasI-RBS-RFP
 +
</p>
 +
<p>
 +
&#9679; pTetR-lasR-pLasR-RBS-CFP
 +
</p>
 +
<p>
 +
&#9679; araC
 +
</p>
 +
<p>
 +
&#9679; pBad-TetR
 +
</p>
 +
<p>
 +
&#9679; pLas-TetO
 +
</p>
 +
<p>
 +
Oscillator Population Tests (population with induction plasmid)
 +
</p>
 +
<p>
 +
&#9679; pop2 0:1 - some fluorescence
 +
</p>
 +
<p>
 +
&#9679; pop2 75:1 - no visual difference
 +
</p>
 +
<p>
 +
&#9679; pop3 0:1 - some fluorescence
 +
</p>
 +
<p>
 +
&#9679; pop3 75:1 - no visual difference
 +
</p>
 +
<p>
 +
&#9679; pop2 and pop3 0:1 - little fluorescence
 +
</p>
 +
</div>
 +
<div id="page36" class='notebook_page'><p>
 +
&#9679; pop2 and pop3 75:1 - a visual increase of both RFP and CFP
 +
</p>
 +
<p>
 +
Plasmid Isolation
 +
</p>
 +
<p>
 +
&#9679; pLac-lasI-RBS-RFP: Concentration: 115.8
 +
</p>
 +
<p>
 +
&#9679; pLasR-LuxI-RBS-YFP: Concentration: 93.1
 +
</p>
 +
<p>
 +
&#9679; pBad-LsrK-plsrA-LuxI-RBS-YFP: Concentration: 128.6
 +
</p>
 +
<p>
 +
&#9679; plac-LuxR-pLuxR-LasI-RBS-RFP: Concentration: 105.9
 +
</p>
 +
<p>
 +
&#9679; pTetR-lasR-pLasR-RBS-CFP: Concentration: 122.3
 +
</p>
 +
<p>
 +
&#9679; araC: Concentration: 114.0
 +
</p>
 +
<p>
 +
&#9679; pBad-TetR: Concentration: 315.1
 +
</p>
 +
<p>
 +
&#9679; pLas-TetO: Concentration: 102.3
 +
</p>
 +
<p>
 +
Digestion and gel extraction
 +
</p>
 +
<p>
 +
&#9679; ad-LsrK-plsrA-LuxI-RBS-YFP: gel not used
 +
</p>
 +
<p>
 +
&#9679; plac-LuxR-pLuxR-LasI-RBS-RFP: gel not used
 +
</p>
 +
<p>
 +
&#9679; pTetR-lasR-pLasR-RBS-CFP: gel not used
 +
</p>
 +
<p>
 +
&#9679; araC: gel not used
 +
</p>
 +
<p>
 +
&#9679; pBad-TetR: gel not used
 +
</p>
 +
</div>
 +
 
 +
 
 +
<div id="page37" class='notebook_page'><p>
 +
&#9679; pLas-TetO: gel not used
 +
</p>
 +
<p>
 +
&#9679; LasR-TT: Concentration: 10.0
 +
</p>
 +
<p>
 +
&#9679; LuxS: Concentration: 3.1
 +
</p>
 +
<p>
 +
&#9679; LuxS: Concentration: 5.5
 +
</p>
 +
<p>
 +
Ligation
 +
</p>
 +
<p>
 +
&#9679; Vector 1: pyear
 +
</p>
 +
<p>
 +
&#9679; Insert 1: LuxS
 +
</p>
 +
<p>
 +
&#9679; Vector 2: pyear
 +
</p>
 +
<p>
 +
&#9679; Insert 2: LuxS
 +
</p>
 +
<p>
 +
&#9679; Vector 3: pTetR
 +
</p>
 +
<p>
 +
&#9679; Insert 3:lasR-TT
 +
</p>
 +
<p>
 +
&#9679; Vector 4: pLac-LuxR
 +
</p>
 +
<p>
 +
&#9679; Insert 4: TT
 +
</p>
 +
<p>
 +
&#9679; Vector 5: pLuxR
 +
</p>
 +
<p>
 +
&#9679; Insert 5: arac
 +
</p>
 +
<p>
 +
&#9679; Vector 6: pBad-TetR
 +
</p>
 +
</div>
 +
 
 +
<div id="page38" class='notebook_page'><p>
 +
&#9679; Insert 6: TT
 +
</p>
 +
<p>
 +
&#9679; Vector 7: plasR-TetO
 +
</p>
 +
<p>
 +
&#9679; Insert 7: RBS-CFP
 +
</p>
 +
<p>
 +
<strong>August 20</strong>
 +
</p>
 +
<p>
 +
Colonies picked
 +
</p>
 +
<p>
 +
&#9679; pyear-LuxR: 1 picked
 +
</p>
 +
<p>
 +
&#9679; pyear-LuxR: 1 picked
 +
</p>
 +
<p>
 +
&#9679; pTet-lasR-TT: 1 picked
 +
</p>
 +
<p>
 +
&#9679; pLac-LuxR-TT: 1 picked
 +
</p>
 +
<p>
 +
&#9679; pBad-TetR-TT: 1 picked
 +
</p>
 +
<p>
 +
&#9679; pLuxR-arac: 1 picked
 +
</p>
 +
<p>
 +
&#9679; pLasR-TetO-RBS-CFP: 1 picked after being repeated
 +
</p>
 +
<p>
 +
Innoculation
 +
</p>
 +
<p>
 +
&#9679; pTetR-LuxI
 +
</p>
 +
<p>
 +
&#9679; pLac-lasI
 +
</p>
 +
<p>
 +
&#9679; pop3 w/TT
 +
</p>
 +
</div>
 +
 
 +
<div id="page39" class='notebook_page'><p>
 +
&#9679; pop2 w/TT
 +
</p>
 +
<p>
 +
&#9679; pop3 w/o TT
 +
</p>
 +
<p>
 +
&#9679; pop2 w/o TT
 +
</p>
 +
<p>
 +
<strong>August 21</strong>
 +
</p>
 +
<p>
 +
Glycerol Stock and plasmid isolation
 +
</p>
 +
<p>
 +
&#9679; pyear-LuxR: Concentration: 40.0
 +
</p>
 +
<p>
 +
&#9679; pyear-LuxR: Concentration: 75.0
 +
</p>
 +
<p>
 +
&#9679; pTet-lasR-T: Concentration: 127.6
 +
</p>
 +
<p>
 +
&#9679; pLac-LuxR-TT: Concentration: 123.5
 +
</p>
 +
<p>
 +
&#9679; pBad-TetR-TT: Concentration: 141.0
 +
</p>
 +
<p>
 +
&#9679; pLuxR-arac: Concentration: 84.0
 +
</p>
 +
<p>
 +
Digestion and gel extraction
 +
</p>
 +
<p>
 +
&#9679; pLasR-LuxI-YFP: gel not used
 +
</p>
 +
<p>
 +
&#9679; pTetR-lasR-TT: gel not used
 +
</p>
 +
<p>
 +
&#9679; pLuxR-araC: gel not used
 +
</p>
 +
<p>
 +
&#9679; pyear-LuxS: gel not used
 +
</p>
 +
</div>
 +
 
 +
<div id="page40" class='notebook_page'><p>
 +
&#9679; pyear-LuxS: gel not used
 +
</p>
 +
<p>
 +
&#9679; pop1 (test)
 +
</p>
 +
<p>
 +
&#9679; pop2 (test)
 +
</p>
 +
<p>
 +
&#9679; pop3 (test)
 +
</p>
 +
<p>
 +
Innoculation
 +
</p>
 +
<p>
 +
&#9679; pyear-LuxS
 +
</p>
 +
<p>
 +
Ligation
 +
</p>
 +
<p>
 +
&#9679; Vector 1: pTet-LasR-TT
 +
</p>
 +
<p>
 +
&#9679; Insert 1: pLasR-LuxI-RBS-YFP
 +
</p>
 +
<p>
 +
&#9679; Vector 2: pLuxR-araC
 +
</p>
 +
<p>
 +
&#9679; Insert 2: lasR-TT
 +
</p>
 +
<p>
 +
<strong>August 22</strong>
 +
</p>
 +
<p>
 +
Digestion (test)
 +
</p>
 +
<p>
 +
&#9679; population 2 (oscillator)
 +
</p>
 +
<p>
 +
&#9675; undigested
 +
</p>
 +
<p>
 +
&#9675; EcoRI
 +
</p>
 +
</div>
 +
 
 +
 
 +
<div id="page41" class='notebook_page'><p>
 +
&#9675; XbaI
 +
</p>
 +
<p>
 +
&#9675; Spe1
 +
</p>
 +
<p>
 +
&#9675; Pst1
 +
</p>
 +
<p>
 +
&#9675; E/P
 +
</p>
 +
<p>
 +
&#9675; X/S
 +
</p>
 +
<p>
 +
Innoculation
 +
</p>
 +
<p>
 +
&#9679; population 1 (oscillator)
 +
</p>
 +
<p>
 +
Colonies picked
 +
</p>
 +
<p>
 +
&#9679; pTetR-lasR-TT-pLasR-LuxI-RBS-YFP
 +
</p>
 +
<p>
 +
&#9679; pLuxR-araC-LasR-TT
 +
</p>
 +
<p>
 +
&#9679; pLasR-TetO-RBS-CFP (2 colonies picked)
 +
</p>
 +
<p>
 +
Transformation
 +
</p>
 +
<p>
 +
&#9679; pLac-LuxR
 +
</p>
 +
<p>
 +
Innoculation
 +
</p>
 +
<p>
 +
&#9679; pLuxR-LasI-RBS-RFP
 +
</p>
 +
<p>
 +
Population Testing Prep (pop2, pop3, pop2+pop3)
 +
</p>
 +
</div>
 +
<div id="page42" class='notebook_page'><p>
 +
&#9679; 2 ml of medium added to each test tube
 +
</p>
 +
<p>
 +
&#9679; 100 microliters of each population added as necessary
 +
</p>
 +
<p>
 +
&#9679; .75mL of inducing population added as necessary
 +
</p>
 +
<p>
 +
&#9675; pop2 w/ w/o inducer
 +
</p>
 +
<p>
 +
&#9675; pop3 w/ w/o inducer
 +
</p>
 +
<p>
 +
&#9675; pop2+pop3 w/ w/o inducer
 +
</p>
 +
<p>
 +
<strong>August 23</strong>
 +
</p>
 +
<p>
 +
Glycerol Stock
 +
</p>
 +
<p>
 +
&#9679; pLasR-TetO-RBS-CFP (X2)
 +
</p>
 +
<p>
 +
&#9679; pTetR-lasR-TT-pLasR-LuxI-RBS-YFP
 +
</p>
 +
<p>
 +
&#9679; pLuxR-araC-LasR-TT
 +
</p>
 +
<p>
 +
Plasmid Isolation
 +
</p>
 +
<p>
 +
&#9679; pLasR-TetO-RBS-CFP: Concentration: 113.7
 +
</p>
 +
<p>
 +
&#9679; pLasR-TetO-RBS-CFP: Concentration: 112.0
 +
</p>
 +
<p>
 +
&#9679; pTetR-lasR-TT-pLasR-LuxI-RBS-YFP: Concentration: 167.0
 +
</p>
 +
<p>
 +
&#9679; pLuxR-araC-LasR-TT: Concentration: 104.2
 +
</p>
 +
</div>
 +
<div id="page43" class='notebook_page'><p>
 +
&#9679; pLac-lasI-RBS-RFP: Concentration: 37.0
 +
</p>
 +
<p>
 +
Colonies picked
 +
</p>
 +
<p>
 +
&#9679; pLac-LuxR
 +
</p>
 +
<p>
 +
Population Testing from 8/22 prep after 24 hours
 +
</p>
 +
<p>
 +
&#9679; 500 microliters from test tube of populations put in microcentrifuge tubes
 +
</p>
 +
<p>
 +
&#9675; pop2 w/: slight increase of RFP
 +
</p>
 +
<p>
 +
&#9675; pop2 w/o: some RFP
 +
</p>
 +
<p>
 +
&#9675; pop3 w/: no significant increase of CFP
 +
</p>
 +
<p>
 +
&#9675; pop3 w/o: some CFP
 +
</p>
 +
<p>
 +
&#9675; pop2+pop3 w/: no significant increase of RFP/CFP
 +
</p>
 +
<p>
 +
&#9675; pop2+pop3 w/o: some RFP/CFP
 +
</p>
 +
<p>
 +
&#9679; Results:
 +
</p>
 +
<p>
 +
&#9675; test tubes were checked after 24 hours
 +
</p>
 +
<p>
 +
&#9632;pop2 showed a slight increase in RFP production when it was induced in comparison to its uninduced control
 +
</p>
 +
<p>
 +
&#9632;both pop3 and pop2+pop3 showed no significant differences between its positive and negative controls
 +
</p>
 +
<p>
 +
&#9675; after 48 hours
 +
</p>
 +
</div>
 +
<div id="page44" class='notebook_page'><p>
 +
&#9632;pop2 showed increase in RFP production
 +
</p>
 +
<p>
 +
&#9632;pop3 showed no significant increase in CFP production
 +
</p>
 +
<p>
 +
&#9632;pop2+pop3 showed a sig increase in RFP production but no CFP when it was induced compared to its negative control
 +
</p>
 +
<p>
 +
Digestion
 +
</p>
 +
<p>
 +
&#9679; plac-LuxR(S/P): Concentration: 19.7
 +
</p>
 +
<p>
 +
&#9679; pTetR-LasR (E/P): Concentration: gel not used
 +
</p>
 +
<p>
 +
&#9679; pLasR-RBS-CFP (X/P): Concentration: gel not used
 +
</p>
 +
<p>
 +
&#9679; pLac-LuxR-TT (S/P): Concentration: 24.0
 +
</p>
 +
<p>
 +
&#9679; pBad-TetR-TT (S/P): Concentration: 15.4
 +
</p>
 +
<p>
 +
&#9679; pTetR-LasR-TT-pLasR-LuxI-RBS-YFP (E/P): Concentration: 12.2
 +
</p>
 +
<p>
 +
&#9679; pLuxR-araC-lasR-TT (X/P): Concentration: gel not used
 +
</p>
 +
<p>
 +
&#9679; pLasR-TetO-RBS-CFP (X/P): Concentration: 5.2
 +
</p>
 +
<p>
 +
&#9679; pLasR-TetO-RBS-CFP (X/P): Concentration: 5.2
 +
</p>
 +
<p>
 +
&#9679; pLuxR-LasI-RBS-RFP (X/P): Concentration: gel not used
 +
</p>
 +
<p>
 +
Innoculation
 +
</p>
 +
<p>
 +
&#9679; pyear-luxS
 +
</p>
 +
</div>
 +
<div id="page45" class='notebook_page'><p>
 +
&#9679; pBad-LsrK-pLsaA-LuxI-RBS-YFP (pop1 oscillator)
 +
</p>
 +
<p>
 +
&#9679; pTetR-LuxI
 +
</p>
 +
<p>
 +
&#9679; pLacI-LuxR-TT-pLuxR-LasI-RBS-RFP (pop2 oscillator)
 +
</p>
 +
<p>
 +
&#9679; pLac-lasI
 +
</p>
 +
<p>
 +
&#9679; pTetR-lasR-TT-pLasR-RBS-CFP (pop3 oscillator)
 +
</p>
 +
<p>
 +
&#9679; pLuxR-lasI-RBS-RFP
 +
</p>
 +
<p>
 +
Ligation
 +
</p>
 +
<p>
 +
&#9679; Vector: pBad-TetR-TT
 +
</p>
 +
<p>
 +
&#9679; Insert: plasR-TetO-RBS-CFP
 +
</p>
 +
<p>
 +
<strong>August 24</strong>
 +
</p>
 +
<p>
 +
Plasmid Isolation
 +
</p>
 +
<p>
 +
&#9679; pluxR-LasI-RBS-RFP: Concentration: 42.6
 +
</p>
 +
<p>
 +
Digestion
 +
</p>
 +
<p>
 +
&#9679; pLuxR-LasI-RBS-RFP (X/P)
 +
</p>
 +
<p>
 +
&#9679; pLuxR-araC-lasR-TT (X/P)
 +
</p>
 +
</div>
 +
<div id="page46" class='notebook_page'><p>
 +
Innoculation
 +
</p>
 +
<p>
 +
&#9679; pLuxR-LasI-RBS-RFP
 +
</p>
 +
<p>
 +
&#9679; pLuxR-araC-LasR-TT
 +
</p>
 +
<p>
 +
&#9679; pLsrA-LuxI-YFP
 +
</p>
 +
<p>
 +
August 25
 +
</p>
 +
<p>
 +
Glycerol Stock
 +
</p>
 +
<p>
 +
&#9679; plac-LuxR
 +
</p>
 +
<p>
 +
&#9679; pBad-TetR-TT-plasR-TetO-RBS-CFP
 +
</p>
 +
<p>
 +
&#9679; pBad-TetR-TT-plasR-TetO-RBS-CFP
 +
</p>
 +
<p>
 +
&#9679; pLuxR-araC-lasR-TT
 +
</p>
 +
<p>
 +
Plasmid Isolation
 +
</p>
 +
<p>
 +
&#9679; pLuxR-LasI-RBS-RFP: Concentration: 89.1
 +
</p>
 +
<p>
 +
&#9679; pLuxR-araC-LasR-TT: Concentration: 152.9
 +
</p>
 +
<p>
 +
&#9679; pBad-TetR-TT-plasR-TetO-RBS-CFP: Concentration: 200.0
 +
</p>
 +
<p>
 +
&#9679; pBad-TetR-TT-plasR-TetO-RBS-CFP: Concentration: 156.0
 +
</p>
 +
</div>
 +
<div id="page47" class='notebook_page'><p>
 +
Digestion and Gel Extraction
 +
</p>
 +
<p>
 +
&#9679; pLuxR-LasI-RBS-RFP: (X/P): Concentration: 9.5
 +
</p>
 +
<p>
 +
&#9679; pLuxR-araC-LasR-TT: (X/P): gel not used
 +
</p>
 +
<p>
 +
&#9679; pBad-TetR-TT-plasR-TetO-RBS-CFP: (X/P): gel not used
 +
</p>
 +
<p>
 +
&#9679; pBad-TetR-TT-plasR-TetO-RBS-CFP: (X/P): gel not used
 +
</p>
 +
<p>
 +
Ligation
 +
</p>
 +
<p>
 +
&#9679; Vector1: pLac-LuxR-TT
 +
</p>
 +
<p>
 +
&#9679; Insert1: pLuxR-LasI-RBS-RFP
 +
</p>
 +
<p>
 +
&#9679; Vector2: pTetR-LasR
 +
</p>
 +
<p>
 +
&#9679; Insert2: pLasR-CFP
 +
</p>
 +
<p>
 +
&#9679; Vector3: pLac-LuxR-TT
 +
</p>
 +
<p>
 +
&#9679; Insert3: pLuxR-LasI-RBS-RFP
 +
</p>
 +
<p>
 +
Primer Dilution
 +
</p>
 +
<p>
 +
&#9679; I3.PsB1A2.R +661ul NFH2O -&gt; 1:10 Dulition
 +
</p>
 +
<p>
 +
&#9679; I3.DT.F +634ul NFH2O -&gt; 1:10 Dulition
 +
</p>
 +
<p>
 +
&#9679; I3.FT.M +700ul NFH2O -&gt; 1:10 Dulition
 +
</p>
 +
</div>
 +
<div id="page48" class='notebook_page'><p>
 +
Colony PCR
 +
</p>
 +
<p>
 +
&#9679; pBad-TetR-TT-plasR-TetO-RBS-CFP
 +
</p>
 +
<p>
 +
&#9679; pLuxR-araC-LasR-TT
 +
</p>
 +
<p>
 +
&#9675; Primer 1: I3.24.SP1
 +
</p>
 +
<p>
 +
&#9675; Primer 2: I3.PSB1A2.R
 +
</p>
 +
<p>
 +
Innoculation
 +
</p>
 +
<p>
 +
&#9679; pyear-luxS
 +
</p>
 +
<p>
 +
&#9679; pBad-LsrK-pLsaA-LuxI-RBS-YFP (pop1 oscillator)
 +
</p>
 +
<p>
 +
&#9679; pTetR-LuxI
 +
</p>
 +
<p>
 +
&#9679; pLacI-LuxR-TT-pLuxR-LasI-RBS-RFP (pop2 oscillator)
 +
</p>
 +
<p>
 +
&#9679; pLac-lasI
 +
</p>
 +
<p>
 +
&#9679; pTetR-lasR-TT-pLasR-RBS-CFP (pop3 oscillator)
 +
</p>
 +
<p>
 +
&#9679; pLuxR-lasI-RBS-RFP
 +
</p>
 +
</div>
 +
<div id="page49" class='notebook_page'><p>
 +
<strong>August 26</strong>
 +
</p>
 +
<p>
 +
Colonies Picked
 +
</p>
 +
<p>
 +
&#9679; pLuxR-araC-LasR-TT: 1 colony picked
 +
</p>
 +
<p>
 +
Population testing
 +
</p>
 +
<p>
 +
&#9679; 2ml broth, 1ml induction, 100ul population, 30 hour incubation
 +
</p>
 +
<p>
 +
&#9675; pop1+2 (+/-): no fluorescence seen
 +
</p>
 +
<p>
 +
&#9675; pop2+3 (+/-): no fluorescence seen
 +
</p>
 +
<p>
 +
&#9675; pop1+2+3 (+/-): no fluorescence seen
 +
</p>
 +
<p>
 +
<strong>August 27</strong>
 +
</p>
 +
<p>
 +
Plasmid Isolation
 +
</p>
 +
<p>
 +
&#9679; pLuxR-araC-LaR-TT: Concentration: 164.3
 +
</p>
 +
<p>
 +
Digestion and Gel extraction
 +
</p>
 +
<p>
 +
&#9679; pLuxR-araC-LaR-TT (X/P): Concentration: 55.1
 +
</p>
 +
<p>
 +
Innoculation
 +
</p>
 +
<p>
 +
&#9679; pyear-luxS
 +
</p>
 +
<p>
 +
&#9679; pBad-LsrK-pLsaA-LuxI-RBS-YFP (pop1 oscillator)
 +
</p>
 +
</div>
 +
<div id="page50" class='notebook_page'><p>
 +
&#9679; pTetR-LuxI
 +
</p>
 +
<p>
 +
&#9679; pLacI-LuxR-TT-pLuxR-LasI-RBS-RFP (pop2 oscillator)
 +
</p>
 +
<p>
 +
&#9679; pLac-lasI
 +
</p>
 +
<p>
 +
&#9679; pTetR-lasR-TT-pLasR-RBS-CFP (pop3 oscillator)
 +
</p>
 +
<p>
 +
&#9679; pLuxR-lasI-RBS-RFP
 +
</p>
 +
<p>
 +
Ligation
 +
</p>
 +
<p>
 +
&#9679; Vector1: pBad-tetR-TT
 +
</p>
 +
<p>
 +
&#9679; Insert1: pLasR-TetO-RBS-CFP
 +
</p>
 +
<p>
 +
&#9675; 0:1 20 colonies seen
 +
</p>
 +
<p>
 +
&#9675; 2:1 50 colonies seen
 +
</p>
 +
<p>
 +
&#9679; Vector2:pLac-LuxR-TT
 +
</p>
 +
<p>
 +
&#9679; Insert2: pLuXR-araC-lasR-TT
 +
</p>
 +
<p>
 +
&#9675; 0:1 5 colonies seen
 +
</p>
 +
<p>
 +
&#9675; 2:1 25 colonies seen
 +
</p>
 +
</div>
 +
<div id="page51" class='notebook_page'><p>
 +
<strong>August 28</strong>
 +
</p>
 +
<p>
 +
Colony PCR
 +
</p>
 +
<p>
 +
&#9679; pBad-tetR-TT-pLasR-TetO-RBS-CFP
 +
</p>
 +
<p>
 +
&#9679; pLac-LuxR-TT- pLuXR-araC-lasR-TT
 +
</p>
 +
<p>
 +
&#9675; Primer 1: I3.24.SP1
 +
</p>
 +
<p>
 +
&#9675; Primer 2: I3.PSB1A2.R
 +
</p>
 +
<p>
 +
Population Testing
 +
</p>
 +
<p>
 +
&#9679; 200ul medium, 200ul induction, 4ul population, 24 hour incubation
 +
</p>
 +
<p>
 +
&#9675; pop1+2 (-): no fluorescence seen
 +
</p>
 +
<p>
 +
&#9675; pop1+2 (+): some sign of RFP/YFP
 +
</p>
 +
<p>
 +
&#9675; pop2+3 (-): no fluorescence seen
 +
</p>
 +
<p>
 +
&#9675; pop2+3 (+): no fluorescence seen
 +
</p>
 +
<p>
 +
&#9675; pop1+2+3 (-): no fluorescence seen
 +
</p>
 +
<p>
 +
&#9675; pop1+2+3 (+): no fluorescence seen
 +
</p>
 +
</div>
 +
<div id="page52" class='notebook_page'><p>
 +
Ligation
 +
</p>
 +
<p>
 +
&#9679; Vector:pLac-LuxR-TT
 +
</p>
 +
<p>
 +
&#9679; Insert: pLuXR-araC-lasR-TT
 +
</p>
 +
<p>
 +
&#9675; 0:1 0 colonies seen
 +
</p>
 +
<p>
 +
&#9675; 2:1 30 colonies seen
 +
</p>
 +
<p>
 +
&#9675; 4:1 15 colonies seen
 +
</p>
 +
<p>
 +
Digestion and Gel Extraction
 +
</p>
 +
<p>
 +
&#9679; pBad-TetR-TT (S/P): Concentration: 88.3
 +
</p>
 +
<p>
 +
Innoculation
 +
</p>
 +
<p>
 +
&#9679; pyear-luxS
 +
</p>
 +
<p>
 +
&#9679; pBad-LsrK-pLsaA-LuxI-RBS-YFP (pop1 oscillator)
 +
</p>
 +
<p>
 +
&#9679; pTetR-LuxI
 +
</p>
 +
<p>
 +
&#9679; pLacI-LuxR-TT-pLuxR-LasI-RBS-RFP (pop2 oscillator)
 +
</p>
 +
<p>
 +
&#9679; pTetR-lasR-TT-pLasR-RBS-CFP (pop3 oscillator)
 +
</p>
 +
</div>
 +
<div id="page53" class='notebook_page'><p>
 +
<strong>August 29</strong>
 +
</p>
 +
<p>
 +
Population Test Prep
 +
</p>
 +
<p>
 +
&#9679; 2ml medium, 400ul populations, 1500ul induction
 +
</p>
 +
<p>
 +
&#9675; pop1+2 (+/-)
 +
</p>
 +
<p>
 +
&#9675; pop2+3 (+/-)
 +
</p>
 +
<p>
 +
&#9675; pop1+2+3 (+/-)
 +
</p>
 +
<p>
 +
&#9679; 1ml medium, 200ul populations, 750ul induction
 +
</p>
 +
<p>
 +
&#9675; pop2+3 (+/-)
 +
</p>
 +
<p>
 +
<strong>August 30</strong>
 +
</p>
 +
<p>
 +
Ligation
 +
</p>
 +
<p>
 +
&#9679; Vector: pBad-TetR-TT
 +
</p>
 +
<p>
 +
&#9679; Insert: plasR-TetO-CFP
 +
</p>
 +
<p>
 +
&#9675; 0:1 100 Colonies seen
 +
</p>
 +
<p>
 +
&#9675; 1:1 200 Colonies seen
 +
</p>
 +
<p>
 +
&#9675; 4:1 200 Colonies seen
 +
</p>
 +
</div>
 +
<div id="page54" class='notebook_page'><p>
 +
Colony PCR
 +
</p>
 +
<p>
 +
&#9679; pLac-LuxR-TT-pLuXR-araC-lasR-TT (X2)
 +
</p>
 +
<p>
 +
&#9675; Primer 1: I3.24.SP1
 +
</p>
 +
<p>
 +
&#9675; Primer 2: I3.PSB1A2.R
 +
</p>
 +
<p>
 +
Colonies picked
 +
</p>
 +
<p>
 +
&#9679; pLac-LuxR-TT-pLuXR-araC-lasR-TT (X2)
 +
</p>
 +
</div>
 +
 
</html>
</html>

Revision as of 22:09, 31 August 2012

Cover page

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)

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