Week 1: June 1-2
Thursday (06/01/17)
Exciting first day orientation and safety training off-Base for the newly assembled crew. The mentors and students bonded as work started towards a common goal.
Friday (06/02/17)
On the second day, the students began learning and overviewing basic lab
techniques. These techniques include: conversion factors, serial dilutions,
micro pipetting, and creating and pouring plates of LB agar. Under the
tender supervision of knowledgable mentors, the students learned to accurately
master lab techniques that would prove invaluable once lab work began.
Week 2: June 5-9
Monday (06/05/17) and Tuesday (06/06/17)
The first two days of this week were spent on Wright Patt Air Force Base
(WPAFB). The team members toured the two labs that they would be
working in and received safety training in both. The student researchers
were very excited to tour the world-class facilities on base. Multiple brainstorming
discussions were also held to finalize the project idea. After the week of training, the
group of students split up into 2 groups: one to work in the 711th Human
Performance Wing, and one to work in the Materials Directorate. The young
researchers at the 711th Human Performance Wing were tasked with sensing and
response while the researchers at the Materials Directorate were tasked with
packaging.
Wednesday (06/07/17)
On the first day in the lab the members made LB broth, (formula in
protocol tab). Although a simple procedure, this was the first hands-on
experience for the students in the lab. They then did a plasma
transformation protocol using E.coli JM109 and the plasmid PRhl_GFPa1. The
bacteria created was plated onto LB chloramphenicol and left in incubator (37°C),
overnight. If there was growth on the plates in the morning there would a greater
probability that the E. coli absorbed the plasmid. The tired but excited young
researchers went home with fingers crossed.
Thursday (06/08/17)
When they entered the lab on the second day the plates showed growth
with distinctions of individual
colonies. Mentor Mike Goodson
ecstatically let the students know about the
significance of the growth and students were
relieved that their plates had grown! These
colonies were used to complete the PCR
protocol. The master mixture, (protocol tab) and
LB broth in round bottom falcon tubes were left
in the incubator (37°C), overnight. The plates
were put in the refrigerator (4°C) to preserve the
bacteria for future use. This protocol was used
to confirm that E.coli JM109 had absorbed the
plasmid PRhl_GFPa1
Friday (06/09/17)
For the last day of the week, the students’ goal was to confirm the E. Coli
had absorbed the plasmid and then perform a MiniPrep Kit to take the
plasmid out of the bacteria. To accomplish this, they first learned how to
create a gel and how to use the electrophoresis. The electrophoresis sends the DNA
down the gel using a positive and negative charge. Under a blacklight you can see if
the DNA is present. Next, the students use a QIA prep spin Miniprep Kit to take
plasmid out of bacteria. The students placed one nanodrop onto spectrophotometer
to measure how pure the DNA extracted is. For future experiments, the plasmid DNA
would then be sent to an offsite lab to be sequenced.
Week 3: June 12-16
Monday (06/12/17)
The students went to a group meeting within their labs. Students
streaked bacteria onto agar plates and put back into the incubator. The
rest of the day was spent collaborating on website design and graphic
design. Two coding novices, Annie and Jason, worked on learning html, CSS, and
Javascript. Graphic designer Tina created a new mascot microbe. Assistant graphic
designer and artist Annie created a drawing of the original Wright Flyer and assisted
in Photoshop drawing. Stenographer Hayley typed up protocols and helped organize
team documents.
The whole team met in the morning to discuss the Wiki and each group’s project.
The RX team observed the preparation and running of PCR. The team made LB
broth and agar. Later the team observed the purification of the PCR results and the
analyzation of the results with Qubit.
Tuesday (06/13/17)
-The students removed the agar plates with bacteria from the incubator.
Two tubes were filled with LB broth, one labeled as a blank and one
containing the bacteria. Using a sterile toothpick, a single colony was picked
from each plate, and placed in the culture tube. An unused sterile toothpick was
added to the blank tube, to verify that the process was sterile. These tubes were
then shaken in an incubator for 3 hours.
The students conducted an experiment using culture growth to see if the bacteria
will turn green with the addition of the quorum sensing molecule to the bacteria.
The next morning, the plasmid should glow green indicating it is present.
Wednesday (06/14/17)
The students retrieved the test tubes placed in the incubator the day before
and analytically checked the optical density and the fluorescence of the
colonies. Immediately afterwards, a visual check was performed as well,
confirming that the Colony + 3OC12 glowed green. The ecstatic students celebrated
as anticipated results showed through the light.
In the afternoon, the students were back at work again preparing a mini-prep on the
PLCD-GFPa1 (the plate streaked on Monday). The remaining DNA containing the
GFP was then placed in the -20o C freezer for storage. Having much more
experience the second time doing the mini-prep, the trailblazing students had a
much better grasp and spent less time with more accuracy.
Thursday (06/15/17)
-Time to go back to work bright and early. A little after the crack of dawn,
the engaged students immediately went to work preparing a digest. The
scholars made a 15 uL digest along with a Master Mix. Interactive steps
included preparing a mini-prep, mixing with enzyme BSr61-HF, enzyme KpuI-HF, buffer Cutsmart, and water. The remaining solution was then placed in incubation for
2 hours. In addition, students were excited to read and learn about plasmid cloning
by restriction enzyme digest and restriction endonuclease. With a desire to learn
more, the students trekked back to the lab and met with another qualified scientist
to assist in the digest. Once the digest was
done, the students made a gel and
electrophoresis to analyze the DNA. The
electrophoresis sends the DNA down the
gel using a positive and negative charge
and allows the students to see the amount
of base pairs, and in this case also allows
the students to see the cut DNA and
replacement. However, the young scientists
were very disappointed to find out the the
digest did not go as expected and DNA did
not get replaced as expected. Disappointed
but not defeated, the students immediately
went back to work and retraced their steps,
beginning the process all over again.
Things do not always work out in the lab
and the students were eager to restart and achieve favorable results.
Friday (06/16/17)
-Immediately the next day, the eager students were right back at work. The
previous day's failure had fired them up and they were eager to do
everything carefully and quickly. The day began with a student-led mini-prep
in which the students were able to follow the protocol and do everything by
themselves. Afterwards, the students and mentors went out for a cool ice cream
treat. All 12 days of hard work deserved a celebration and the students and mentors
bonded over frozen custard and ice cream.
