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pGlo Lab Report

Purpose:
The purpose of this lab was to learn about genetically modified organisms. To do
this we transferred the pGlo gene to E.coli to genetically modify the bacteria to make it
glow.

Hypothesis:
We believe that there will be growth on all of the +DNA plates and the LB −DNA
plate because there is nothing preventing the bacteria’s growth on any of the plates. We
think that there will be no bacteria colonies on the LB/ amp −DNA plate because that
bacteria are not resistant to ampicillin. And finally, we believe the only plate that will
glow is the LB/amp/ara plate because that one has the protein that will activate the
glowing trait in the E.coli.

Procedure:
For this lab we used the Babec pGlo transformation instructions.
“pGlo Transformation.” ​BABEC: Bay Area Bioscience Education Community​,
babec.org/gfp-transformation/.

Data:

+DNA on LB plate −DNA on LB plate


→Many small colonies →Large number of colonies, bigger in size
+DNA on LB/amp plate −DNA on LB/amp plate
→A few colonies, big in size →Yellow circle shows the contamination we
had

+DNA on LB/amp/ ara plate


→The colonies glowed slightly green, but not very strong

Analysis:
We got growth on all the plates we thought we would. We got growth on the LB
plates because those plates were designed to have the bacteria grow. LB stands for
Luria Broth, which grows the bacteria and helps the E.coli recover after the various
temperature shocks. We had growth on the LB/ampicillin +DNA plate and no growth on
the LB/ampicillin −DNA plate. This was because the +DNA had the pGlo gene which is
resistant to the ampicillin which was in that plate. Ampicillin is basically a different strain
of penicillin that would kill the E.coli when it does not have the pGlo gene. The −DNA
had no pGlo meaning it was not immune to the ampicillin, which meant that there should
have been no growth on the plate. The final plate had LB/amp/ara which should activate
the protein that would make the bacteria glow. Ara stands for the sugar arabinose,
which turns on the pGlo during transcription. Our plate did glow, but not too strong,
meaning we could have made a mistake.
Our hypothesis proved to be partially right, because our LB/amp/ara plate did not
glow to strong, but it was glowing.
Our mistake was probably a result of our timing. We left our +DNA in the ice bath
before the heat shock a little too long. In the heat shock the water was also not hot
enough. Both of those to factor could have resulted in the bacteria not glowing as bright
because those two things had to be very precise and our temperatures and times were
not as accurate. I think it was a mistake in one or both of those areas because the
bacteria would have probably still glowed, but not as bright.
We could have improved this lab if we had more time in class so we were not
rushing through the last few steps which could have caused a mistake in the results. We
could have also made sure the water was heated up beforehand so that some of the
DNA would not go into the heat shock when it wasn’t warm enough or that some of us
left the DNA in the ice for too long waiting for the water to heat up.
This lab had left me curious about how you would change the color of the pGlo
gene, so you can express other colors in the bacteria. Also, I wonder how you can make
other organisms glow and what the process would be for that.

Conclusion:
We learned that you can transfer genes to different organism. To learn how to
transfer genes, we grew E.coli on various plate that would make it glow when the gene
pGlo was added. In order to make it glow we had to do various temperature shocks to
the bacteria and had to feed it. We discovered this by having a control −DNA to see if
we would have growth. This would tell us if we did the lab correctly because all of the
bacteria should grow on the LB plate. The −DNA should have not grown on the LB/amp
plate because we never put any of the pGlo in the negative DNA which means that it is
not immune to the ampicillin in the plate. Our results proved to follow this pattern, where
we had multiple colonies on the −DNA LB plate and no colonies on the −DNA LB/amp
plate. We had all three +DNA plates because without them we would not have known if
we did the transformation correctly. The LB +DNA plate showed that our E.coli grew, the
LB/amp plate shows if we correctly added the pGlo because if we didn’t then the E.coli
would have not grown since they would not be immune to the ampicillin in the plate.
Finally, we had the LB/amp/ara plate for just the positive because the ara sugar
activates the pGlo in the bacteria to make it glow. All of our positive DNA plates worked
as we thought it would, meaning that the lab worked correctly. We did not make a
negative LB/amp/ara DNA plate because it does not have the pGlo gene, so there
wouldn’t have been any growth, let alone any glowing.