First day in the lab! (After a cancelled meeting due to a typhoon day!)
After researching all of the second semester and summer and coming up with a general protocol, we were ready to begin our whole experimenting process. We caught up on everything that we had done (as all of us hadn’t seen each other throughout the whole summer due to a lot of trips).
During the summer we had decided to use three Anderson promoters due to the variety of strengths they come in and versatility (as they are able to be used in a lot of different situations.
We came up with three strengths of promoters: weak, medium, and strong
Our weak promoter had a strength of 0.33 (BBa_J23110)
Our medium promoter had a strength of 0.58 (BBa_J23111)
Our strong promoter had a strength of 1 (BBa_J23100)
We had also decided to use the C-Osmy tag because we knew that it would be the tag with the best fit for western blot (to track). However we realized later one that we wouldn’t have time for that.
For our N-terminus end we had decided to use myc-tag.
Although no real lab work was done a few of us got together to discuss what vector and linearized backbone we would use
Chose to use pSB1C3 (found in the iGem catalog) for our Gibson assembly as it was already in the distribution kit and would work with our project
We started and finished most of the competent cell test for our e. Coli cells to check its efficiency. We chose to do this before our whole experiment began to make sure that we were using working e. Coli cells.
So we completed that lab up until the final step, which was to count the number of colonies that would grow.
August 25th 2016 - Competent Cell Check/PCR
We first counted our colonies from the competent cell check the day before to check on e.Coli growth
Counting colonies/E.Coli growth
L = Large M = Medium S = Small
Concentration
#1 Plate
#2 Plate
#3 Plate
0.5
0
0
0
5
0
0
0
10
3L 3XL 1R
1S 1XL
2S 2L 1XL
20
36S 3M 7L
10M 1R
8S 4M 3L 1R
50
39S 23M 4L
117S 3M 1L
17S 16M 6L
Area calculations : 10
Extra Large: diameter = 6.5 mm, total area = 99.55 mm^2
Large: diameter = 4.5 mm, total area = 63.62 mm^2
Small: Small = 3 mm, total area = 42.4 mm^2
Rim: Length = 2 cm
Area calculations: 20
Large: diameter = 5 mm, total area = 196.35 mm^2
Medium: diameter = 3.4 mm, total area = 63.55 mm^2
Small: Diameter = 2 mm, total area = 138.23 mm^2
Rim: Diameter = 10.5 cm
Area calculations: 50
Large: Diameter = 5 mm, total area = 215.98 mm^2
Medium: Diameter = 4mm, total area = 527.79 mm^2
Small: Diameter = 2 mm, total area = 543.5 mm^2
Rim: Length = 0
Decided to wait until the next day to finalize our decision on whether or not we want to use the 20 pgp or the 50 pgp with our colonies
There were advantages with both to consider
The 20 pgp had larger colonies while the 50 pgp had more colonies though they were smaller in size
But 0.5 and 10 pgp had been eliminated for sure due to poor growth
Ended up using the 20 pgp because we didn’t want so many small colonies and we didn’t want to use the pgp to be too strong
Following this we PCRed our promoters
Had a little issue with the buffer in that we didn't dilute it but managed to finish replicating all our promoters and the buffer
So the PCR ended up being totally fine as we successfully were able to replicate all the promoters and we were able to recover our buffer back
Decided to wait till the next day to finalize our decision on whether or not we want to use the 20 pgp (?) or the 50 pgp there are advantages with both
The 20 pgp had larger colonies while the 50 pgp had more colonies though they were smaller
But 0.5 and 10 pgp had been eliminated for sure
PCRed our promoters
Had a little issue with the buffer in that we didn't dilute it but managed to finish replicating all our promoters and the buffer
August 26, 2016 - Gibson Assembly
Used the Gibson Assembly to construct the plasmid containing the strongest promoter. Did not include tag.
Transformed the assembled plasmid into E. Coli, then spread onto agar plate and left to grow.
Acted as test run to see if we could successfully construct + transform plasmid.
Fragment Volumes
BCU
PET
OSMY
Promoter
4 ul
4 ul
2 ul
2 ul
Prepared the PET plastic film that our e. Coli will “eat” or degrade
We will be using plastic from a PET bottle called いろいろい (iroiroi)
Used the Gibson Assembly to construct the plasmids containing the medium and weak promoters. Did not include tag.
Transformed the assembled plasmid into E. Coli, then spread onto agar plate and left to grow.
Didn’t put in the MYC tag because we are currently planning on putting it on later, to when we use electrophoresis to see how well the enzyme can be signalled out
August 29, 2016
Started the PCR process
Used a process that our advisor gave to us which can be found underneath the protocol
We decided to PCR the promoters and buffer (for Gibson Assembly) to make sure that we wouldn’t run out of promoters (since they are crucial to our experiment)
Ran the PCR for 30 cycles to clone enough parts
September 2nd 2016
We transferred E. Coli constructs to liquid growth medium (LB medium) so that they would have nutrients to grow
Using a sterile inoculating loop, colonies were transferred from respective plates to the liquid growth medium tubes
Each tube was filled to 30 mL of LB liquid prepared by Ms. Crissy (one of our advisors)—one for control, one for moderately strong promoter, one for weak promoter, one for strongest promoter
Incubating at 37 ºC; started incubating at about 4 pm
September 5, 2016
Used the spectrometer to measure the optical density of the e. Coli cells in our tubes with the LB liquid to see how much they had grown in the LB broth as these would be the cells that we would use
The spectrometer was set at 600 nanometers
Here are the results (the peak absorbance of the light):
Control: 0.851 λ
Strong promoter: 0.965 λ
Medium promoter: 0.900 λ
Weak promoter: 0.832 λ
These are the results we wanted. We were hoping for a figure around 0.800-0.900 that would increase as the promoter got stronger. Thus we now know that our cells in the LB liquid had been growing just fine.
September 9th 2016
Transferring E. Coli constructs to liquid growth medium (LB medium)
Using a sterile inoculating loop, colonies were transferred from respective plates to the liquid growth medium tubes
Each tube was filled to 30 mL of LB liquid prepared by Crissy—one for control, one for moderately strong promoter, one for weak promoter, one for strongest promoter
Incubating at 37 ºC; started incubating at about 4 pm
September 10, 2016
Had to revive our e. Coli colonies in the LB liquid since we hadn’t actually expanded them for about five days
Thus our next step was to actually make sure that they had enough oxygen and LB broth afterwards
We learned from our mistake and decided to set up a sort of 3-day rotation schedule to feed them
Checked on all of our e. Coli colonies to make sure that they grow for sure before we officially begin the experiment with the PET plastic.
We measured the colonies based on their size/areas in mm
The control:
Colony 1: 20 mm
Strong Promoter
Colony 1: 78.5 mm2
Colony 2: 50.24 mm2
Colony 3: 78.5 mm2
Colony 4: 120 mm2
Colony 5: 50 mm2
Medium promoter:
Colony 1: 624 mm2
September 14th 2016
Expanded liquid LB so that we don’t have another round of dying e. Coli cells and can keep a stock just in case
September 16, 2016
Checked up on our e. Coli that are degrading the PET plastic
September 17, 2016
Took PET film mass measurements on Trial 1
Consistency issues in data, gains in mass a problem
Mass measurement procedure:
1. Taken from tray, put on scale
2. Measure and record mass
3. Put it into its appropriate E. Coli suspension
It’s suspected that our stock, because it wasn’t initially maintained well, produce E. Coli that are dying, so we will replace our stock on Monday
We should do Western Blots to check for PETase in supernatant
In a few days we need to create a myc-tagged assembly (Gibson assembly), perform transformations for new plates — ultimately for new stock suspensions — and, once all that is done and we have new stock, start trial 2 with new PET and fresh E. Coli
Transformed the competent e. Coli cells and inserted the myc c-terminus tag
We prepared it up until the point before we transferred it onto the agar plate as it was Back-to-School night and we didn’t want to stink up the room for the parents (we’re so kind)
The cells were thus transferred at night, and so we won’t be able to work with them on the next day to make sure that they transform completely
September 23, 2016
Today we’re taking mass measurement of PET film to check up on the progress and results of our experiments
Poured out the mass e. Coli liquids
Began to clean up the equipment, which required HCl (hydrochloric acid)
