Team:The Agency Escondido/project/scissors

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.

Scissors

The "scissors" bacteria strain will have an RFP (part E1010)and promoter (part I12006). It will contain the AraC gene (part C0080) to induce the "rock" strain.

Notebook

May 20

Vansh Singh began a protocol to prepare competent cells. This protocol omitted the final step of resuspension. Vansh caught this mistake in the protocol and continued the process of making competent cells from the protocol's source.

May 21

Vansh Singh completed cell competency from yesterday.

Vansh Singh resuspended BBa_B0015, BBa_I12006, BBa_I13502,pSBIA3, and pSBICK3. He transformed these parts into competent cells. Unfortunately, a thermocycler program which was supposed to hold parts at 42 degrees Celsius for two minutes went up to 92 degrees Celsius. He stopped the program as it was climbing up to 92 degrees when it was at 90 degrees. Transformation was compromised. However, Vansh Singh continued the transformation process of those cells. Afterwards, he also found it fit to start another round of transformation. Vansh Singh inoculated the transformed cells from his first batch onto an agar plate. However, he had forgotten to add the respective antibiotics to the plates beforehand. Therefore the plates had to be disposed.

Vansh Singh moved the cells into the fridge before he left

May 22

Vansh Singh superficially added Ampicillin to an agar plate and added Kanamayacin to another agar plate. Because of limited plates, he divided each plate into fifths and inoculated cells from each batch into its respective agar plate in its respective "fifth." Vansh Singh made LB agar.

May 23

Vansh Singh inoculated tubes with BBa_B0015, BBa_I12006, BBa_I13502, pSBIA3, and pSBIK3. Hours later, he realized that he had forgot to add antibiotics into the LB in each tube.

May 24

Vansh Singh inoculated new tubes with BBa_B0015, BBa_I12006, pSBIA3, BBa_I13502, IpSBIK3, BBa_K228032, and BBa_K093006 with LB and respective antibiotics.

May 27

Vansh Singh made 1 mL of glycerol stock from parts BBa_B0015, BBa_I12006, pSBIA3, pSBIK3, BBa_K228032, BBa_K093006, and BBa_I13502 which had been grown in selective media over the weekend.

Vansh Singh performed DNA miniprep of the seven parts according to the Promega PureYield(TM) Plasmid Miniprep System.

May 28

Vansh Singh performed Restriction Digest of Assembly 1 of the "Scissors" strain. Evan Santos performed Ligation of Assembly 1 of the "Scissors" strain

May 29

Vansh Singh made competent cells and transformed the ligated plasmid from yesterday into the competent cells. He then inoculated chloremphenicol plates with some cells. The remaining 1ml of cells were stored in the fridge.

May 30

Vansh Singh's plate showed no growth. He inoculated another chloremphenicol plate with the transformed cells.

May 31

Vansh Singh's plates showed no growth.

June 3

Vansh Singh doubted his ligation and ligated another batch of Part X plasmids.

June 4

Vansh Singh and Evan Santos started the protocol for calcium chloride competent cells. The cells were incubated on ice over the night.

June 5

Vansh Singh and Evan Santos finished the calcium chloride competent cell protocol and transformed Part X with the competent cells. Because of the lack of LB agar plates, he left a 1.5ml eppendorf tube with the transformed cells and LB + chloremphenicol in the dry bath set at 37 degrees Celsius.

June 6

Vansh Singh inoculated a 5ml tube of LB media + chloremphenicol from his eppendorf tube. We still lacked chloremphenicol agar plates.

June 7

Yesterday's chloremphenicol tube showed significant cloudiness. He believes that he successfully assembled Part X. Mentor Andrew Segina made 4 LB Agar + kanamayacin plates. Unfortunately Vansh needed chloremphenicol plates. So, Vansh made inoculated a new tube of LB + chloremphenicol from the tube from yesterday.

June 10

Vansh Singh made 4 LB Agar + chloremphenicol plates. He inoculated a plate from his "Part X cells 6/6/13" tube.

June 11

Vansh Singh believed that he saw a colony on his Part X plate. So, he inoculated a flask (We were out of clean tubes) of 10mL LB + Chloremphenicol with the colony on the Part X plate. A single clonal line of Assembly one has successfully been grown.

June 12

Vansh Singh miniprepped Part X according to the Promega PureYield(TM) Plasmid Miniprep System.

June 13

Vansh Singh completed the digestion and ligation of Assemly 2