Team:The Agency Escondido/project/rock

Project Description

Our project seeks to create two identical triads of transparent bacteria strains with complementary hydrophobic inducer production to emulate the game of "rock, paper, scissors" (Hydrophobic proteins can move through plasma membranes). Each triad will correspond to a group: group A and group B. Each type of bacteria will produce a specific protein that will act as an inducer to one and only one other bacteria strain(rock strain, paper strain, and scissors strain). Each triad will have different reporter genes designating its champion color. When two bacteria strains are combined in an agar plate, each protein secreted by the victorious strain, when acting as an inducer, changes the color of the cells on the plate identifying the champion.

Rock

The "rock" bacteria strain will have a CFP (part E0020) and promoter (part R0080). It will contain the LuxR gene (part C0062) to induce the "paper" strain.

Notebook

May 20

Andrew Buss began a protocol to prepare competent cells. This protocol omitted the final step of resuspension.

May 21

Andrew Buss completed the cell competency protocol from the previous day, spinning down 8 mL total in sequence.

With the arrival of ampicillin, Andrew Buss streaked BBa_S01022 onto a quadrant of an ampicillin agar plate.

Andrew Buss began transforming DNA for later assemblies into cells, using 5 uL of resuspended DNA per sample in water as suggested by the Parts Registry protocol. If the transformation failed with 5 uL, then 40 uL containing 1 ug could be used in another protocol. 50 pg of DNA from the transformation efficiency kit was used as an RFP control. The second and last centrifugation in the protocol was carried out using 0.6 mL tubes. Unfortunately, the rotor in the centrifuge was designed for 1.5 mL tubes. When centrifuging, three of the tubes were forced open, and fell through the holes in the rotor. These three samples were lost.

Andrew Buss restarted transformation, using the same protocol. Cells were left to incubate overnight in LB containing the appropriate antibiotics: the transformation control and pSB1C3 used chloramphenicol, while the remainder used ampicillin.

May 22

The chloramphenicol plate from the previous day displayed faint streaks (possibly simply indentations in the agar) but no distinct colonies. The ampicillin plate containing BBa_S01022 displayed dense growth. Andrew Buss extended some streaks onto the next quadrant to reduce the density to individual cells.

Andrew Buss streaked two quadrants of an ampicillin plate with BBa_R0080 and BBa_B0010. BBa_J37033 was transformed on the previous day, but was accidentally omitted from the streaking step.

May 23

The chloramphenicol plate containing BBa_S01022 displayed additional dense growth. Andrew Buss streaked the second half of the plate more thoroughly, attempting to dilute the colonies enough to pick out a single one the next day.

The ampicillin plates and the pSB1C3 quadrant still displayed no growth. Andrew Buss ran a third transformation, using the previous day's NEB10 cells. The competency procedure went well - resuspension occurred readily, and the initial OD 600 of the samples was almost exactly the recommended 0.6.

May 24

Andrew Buss inoculated one tube of ampicillin LB broth with BBa_S01022, and another with BBa_R0080. Other samples did not show convincing colonies.

May 27

Parts BBa_S01022 and BBa_R0080 grew well in selective ampicillin media over the weekend. Andrew Buss prepared glycerol stocks of each and then performed miniprep on both.

Part BBa_37033 was not streaked onto a plate on the 24th, so Andrew Buss streaked the three transformation samples onto quadrants of ampicillin agar prepared on the 23rd.

The chloramphenicol plate containing the transformation control and pSB1C3 displayed growth of red cells inside the pSB1C3 quadrant. Evidently, Andrew Buss had streaked onto the wrong quadrants on the 23rd. An additional quadrant of the plate was streaked from the "221" and "121" samples from the 22nd and 23rd.

Andrew Buss streaked a third quadrant of the chloramphenicol plate with three transformations of pSB1C3.

May 28

The pSB1C3 quadrant showed no growth, nor did the three quadrants of BBa_37033. Another transformation may be attempted the next day if material remains.

Andrew Buss inoculated two tubes of LB media with NEB10 cells for transformation the next day.

Andrew Buss prepared 200 mL of LB media.

May 29

The pSB1C3 quadrant and the three BBa_37033 quadrants showed no growth. Andrew Buss inoculated selective ampicillin media with the entirety of the three transformation outputs. pSB1C3 should have been processed similarly but was omitted by accident.

May 30

The tube containing BBa_37033 showed growth. Andrew Buss inoculated a new tube with the most optically dense transformation, sample 221 of pSB1C3.

May 31

The tube containing sample 221 of pSB1C3 showed no growth. Andrew Buss inoculated two new tubes of chloramphenicol media with samples 121 and 11 of pSB1C3.

Andrew Buss prepared a glycerol stock of BBa_37033.

June 3

Andrew Segina prepped plasmid DNA from pSB1C3 11; J37033; and B0010.

Sample 11 of the pSB1C3 transformation grew well in chloramphenicol media over the weekend. Andrew Buss forgot to create a glycerol stock before miniprep, but, luckily, only a few mL were used for miniprep. A glycerol stock will be created the next day.

Andrew Buss inoculated selective ampicillin media with 500 uL of the BBa_37033 glycerol stock from Friday to check that cells would survive the freezing and thawing process. After several hours, the tube showed growth.

Andrew Buss performed a restriction digest using BBa_R0080 as the upstream part, BBa_S01022 as the downstream part, and pSB1C3 as the destination plasmid, using the NEB protocol for restriction with its BioBrick Assembly kit. Without any means of quantifying the miniprepped DNA from 5/27, Andrew Buss assumed a 50 ng/uL concentration for both samples, based very roughly on estimates of other teams' measurements. Because miniprep took place during the restriction digest, he used the 25 ng/uL pSB1C3 DNA sample from the iGEM 3A Assembly kit.

Andrew Buss completed ligation of the restriction digests. While he had intended to transform immediately, Evan Santos' cell competency protocol went awry, and competent cells were not available. Transformation should take place during the next day.

June 4

Andrew Buss prepared a glycerol stock of pSB1C3 for long term storage from sample 11.

Andrew Buss prepared competent cells using revision 30 of the cell competency protocol by Evan Santos.

Andrew Buss transformed the sample of BBa_S05132 ligated the previous day (sample 28) into the previously prepared competent cells, using revision 5 of the calcium chloride transformation protocol by Vansh Singh. Additionally, 10 uL of 20 pg/uL RFP control was used as a transformation control. After 1 hour of growth in NEB SOC media, the cells were applied to chloramphenicol plates included with the 3A Assembly kit, and spread around with glass beads.

June 5

Vansh inoculated tubes with 5ml LB + chloremphenicol of the two plates from yesterday.

June 6

Yesterday's tubes were kept in the refrigerator, so they did not grow. Andrew Buss returned them to the dry bath.

June 7

The tube containing the transformation control showed excellent growth in chloramphenicol media. However, BBa_S05132 showed no growth. Andrew Buss inoculated another tube of chloramphenicol LB media with a colony from the plate.

June 10

None of Andrew Buss's BBa_S05132 tubes grew. Vansh believed that the fault was in Andrew's assembly. So, Vansh led Evan Santos and Blaise Develasco in restriction digest and ligation of Assembly 1 Rock Protocol.

June 11

Evan Santos, Grant Jose, and Blaise Develasco prepared competent cells.

June 12

Vansh transformed BBa_S05132 and grew a chloremphenicol LB media tube.

June 13

Vansh made inoculated a chloremphenicol plate of BBa_S05132.

June 14

Vansh inoculated a new LB media + chloremphenicol tube with a single colony of BBa_S05132.