Team:Consort Alberta/notebook
Notebook
Jan 18th: Workshop in Lethbridge. Took notes on procedures and definitions. We determined steps for our project: Transformation, grow overnight cultures, miniprep, restriction digest, ligation, transformation.
May 19: Grew JM109 cells overnight.
May 20: Transformed DNA into JM109 cells after making competent cells. Let grow overnight.
May 21: Miniprepped AmilCP and ECOS in JM109
May 22: Restriction digest of ECOS and AmilCP. Puc57 was cut with E and S. Amil CP in PSB1C3 was cut with E and X. cutsmart was used for the buffer.
May 25: Started JM109 cells. Grew for three hours while ligated parts together and transformed into JM109. OD was .51
May 26th: Running a gel to see our results. Labelling system as follows: Plate/tube #/U or B (uncolored colony or blue colony). Gel run as follows
Well 1) Amil CP 1
Well 2) Amil CP2
Well 3) 2/1/U
Well 4) 2/2/U
Well 5)1/1/U
Well 6) 1/2/U
Well 7)1/1/B
Well 8) 1/2/B
Well 9) 2/1/B
Well 10) 2/2/B
August 14th: We were unsure what happened at this point with the coloured and uncoloured culture so we decided to start from the beginning and see what happens. We grew overnight cultures of AmilCP and JM109. They grew well.
August 16th: Miniprepped the Amil CP and stored it in the freezer. Made plates with 4% alginate to make sure we can grow cultures on it.
August 17th: Mini prepped the old version of ECOS (We used ECOS from plates we had in the fridge furthermore referred to as old ECOS). Made competent cells from JM109 and transformed new ECOS (we transformed DNA from the freezer. This batch is refereed to furthermore as new ECOS). Cells were plated overnight. We also made alginate plates. One was a negative control and the other with JM109 cells.
August 18th: Took plates from incubator and started broth with new ECOS. Did restriction digest of AmilCP and old ECOS. We did two different trials. Note that we did both trials with old and new ECOS.
Trial A- puc57 is cut with x and p. was used for buffer. PSB1C3 was cut with E and P. 3.1 was used for the buffer.
Trial B- Puc57 was cut with E and S. Amil CP in PSB1C3 was cut with E and X. cutsmart was used for the buffer.
Labelling system as follows: type.tube# trial letter Ex. 1.1 A
1=AmilCP
2=ECOS old
3= ECOS new
4=PSB1C3
We then ligated our parts together for each of our respective trials. (Old ECOS)
Made competent cells- .5 OD and transformed our ligation into our cells. We ended up with 6 plates. 2 negative controls on amp plates and 4 plates on chlor- two plates from each trial with one 20 micro litre and one 200 micro litre of broth.
August 19th: Made LB cultures of JM109. Miniprepped New ECOS. Made Chlor. plates. Restriction digest of New ECOS. Ligation of New ECOS (using trial A and trial B from yesterday). Made competent cells- .4OD transformed them and grew overnight. We also ran a Gel of all our restriction digests.
ladder
1.1
1.2
1.3
1.4
2.1
2.2
3.1
3.2
3.1
3.2
4.2
Ladder
Our results were as followed:
As you can see the PSB1C3 was the farthest ahead which is what we want as it was the smallest part. All four of our new ECOS were triple banded. We are unsure why this is happening. As we were expecting two bands- one with ECOS and one with the backbone. But AmilCP is between the new ECOS and the PSB1C3 which is what was expected.
Aug 20th: New ECOS transformation results- Dark and light colonies on Trial B 200 micro litres and no growth on 20 micro litres. Some colonies on Trial A 200 micro litres and no growth on 20 micro litres.
Aug 26th: Grew overnight cultures of plates from New ECOS trial A and B.
Aug 27th: Labelling: Trial plate- colouring number (Originals: A1 A2 B1-D B2-D B1-L B2-L) L= light D=Dark (Example: B1-D1). We started a mini-prep but accidentally threw out supernatant we needed. Grew more LB cultures.
Aug 28th: Plate one: negative control. Plate two: 10 microlitres. Plate three: 100 Microlitres. Plate four: 250 microlitres.
tube one: negative control. tube two: 1 microlitre. tube three: 25 microlitres. Tube four: 500 microlitres.
We did a miniprep and restriction digest with ecoR1 and cutsmart on all trials. Ran gel with the following order.
Ladder
A1
A2
B1D
B2D
B1L
B2L
PCB1C3
Ladder
Results as follows:
We also started our experiment. Created plates and tubes and let grow for four hours. Added xylene to tubes. We let the plates grow longer but ended up with overgrowth. We'll have to regrow plates and try again. Tried making alginate beads with Peking protocol. Didn't form beads. Will try again later.
Aug 29th: Today we made chloraphenicol plates. We did more alginate trials-experimenting with [CaCl2], temperature, speed and amounts of alginate. We found we could only make beads with straight LB and forming them with molds. The Peking protocol did not produce beads for us. We also got results from LB trials. When we came in the LB looked totally normal. We couldn’t see any difference. After spinning the colonies down, we did get results. Please refer to the lab write up to see analysis and pics. As we couldn’t see amilCP in broth; doubt the alginate beads will work. They are totally opaque. We also made more colonies for further testing.
Aug 31st: Regrew light one and light 2. Redid lab. Grew light three and four for three hours. Added xylene to all. Light one and two we believe overgrew and died as they were left in the incubator for a day and a half. No results with the xylene. They all look like the negative control.
Sept 5th: Made overnight cultures and plates to use, cleaned up the lab and made plates. Took 20uL of broth and poured it onto 8 plates C4 from tube 1,4 rom tube 2) and 100uL of broth and poured it into 8 plates C4 from tube 1 and 4 from tube 2) Worked on design for poster. Took 9mL LB broth and added 1mL of cell broth in falcon tube to use for overnight cultures C4 overnight cultures C2 from tube 1 and 4 from tube 2)
Sept. 6th: Plates- From our two LB tubes, we made 8 plates each. 4-20uL per tube and 4-100uL per tube.
Plate one- Control
Plate two- 10ul xylene
Plate three- 100uL xylene
Plate four- 50 uL of xylene.
Pipetted all of the xylene onto each plate at their respective amounts while in the fumehood.
Tubes- Take overnight cultures. Pour 9mL of LB broth into 15mL falcon tubes. Take 1mL of overnight cultures and insert it into the second batch of tubes.
Tube 1- control
Tube 2- .5 mL xylene
Tube 3- 10uL xylene
Tube 4- 50uL xylene
Sept 7th: Went to look at results. Plates did not grow. Positive results from all LB tubes. See lab for write up.
Sept 8th: Inoculated colonies to send to parts registry.
Sept. 9th: Mini-prepped cell culture for submission to Parts Registry/sent part away to Registry.
Sept. 16th: Inoculated LB with our ECOS part for experiment "real life" trials with built protoype.
Sept. 17th: In order to prepare for our "real life" experiments, we mixed 3.5mL of xylene for 350mL of soil. We then added the soil to our built prototype and added LB and cell cultures from yesterday to the chamber that holds ECOS. We then let it sit overnight, starting at 4 p.m. on the shaker table.
