Team:Alma/Engineering
Engineering
Improvement of Part: K123002
Our Goal of this Improvement:
This part was our TetR gene, and Red Fluorescent Protein (RFP) gene.
This part is arguably the most important part of our project as it will be
the piece that allows for quantification of the response to DDT that our
biosensor will exhibit. The research conducted with this part of our project
largely focused on the response to anhydrotetracycline (henceforth ATC).
This was used as an analog to the response that would be exhibited by the
estrogen receptor in causing the expression of our TetR gene. By exposing
our bacteria with the K123002 part present to ATC in varying concentrations,
we were able to establish a zone of inhibition that would then allow us to
familiarize ourselves with the reactions of our circuit to the presence of DDT.
The Process:
Originally we received some results that made us think the plasmid backbone was
closing in on itself. We conducted a Gibson assembly with K123002 attempting to have
an “overkill”, or more than needed amount, of insert to see if it would stop the backbone
from closing on itself. We tried using 1 uL of backbone, 2 uL of insert (instead of 1 uL),
10 uL of HiFi Master Mix, and 7 uL of H20.
When we came in to look at the colonies in the morning,
there were none. We let them incubate for longer with monitoring.
We also looked into the possibility of better optimizing this in hopes
of it working, and trying competent cells that have been ordered as
opposed to those made here in our lab.
We then ran a colony PCR for K123002. The forward and reverse
primers used were TET SEQ F2 and TET SEQ R2. When inputting the primer
sequences on the NEB Tm Calculator, it suggested we ran the PCR at 72 C,
which we did. When the gel came out, it was unsuccessful as nothing was
on it. We conclude that it is common for colony PCR to be finicky. We
also ran a plasmid miniprep with 6 colonies off of our K123002 plate.
We then mixed them with a primer and took them down to MSU for Sanger
Sequencing.
We then did a PCR of K123002 (E). The colonies were then harvested and added,
with the PCR being conducted following a 57C annealing and 90S extension protocol.
When the PCR was finished, we ran a gel which gave us seven lanes of results. But,
by appearance, the bacteria colonies did not seem to have uptaken the K123002
BioBrick part as we would expect much larger pieces of DNA to be visible on the gel.
We then did another PCR with 1 uL of backbone and 1 uL of insert.
For reactions with 1 insert (there will be 8 uL of water in these reactions).
For reactions with 2 inserts we will do 1 uL of the backbone and 1 ul of EACH
insert (2 uL insert total) and 7 uL of water.
We ran a K123003 PCR again. These are the primer concentrations used:
The PCR tube 9 had its cap come off during it. It was missing a lot of liquid. More than likely it did not work because of this. Regardless, 10 uL of water was added to check, and it has been run through a gel to see if it could possibly work.
The PCR tube 9 had its cap come off during it. It was missing a lot of liquid. More than likely it did not work because of this. Regardless, 10 uL of water was added to check, and it has been run through a gel to see if it could possibly work.
Lanes 2 and 9 should yield identical results since they are the same, as should lanes
4 and 10, which they did. All four were relatively equal to each other as well. Lane 5
did not seem to work. We continued to do a Gibson Assembly with Lane 2 (Tube 1), Lane 4
(Tube 3), Lanes 3 (Tube 2) and 7 (Tube 6), and Lanes 3 (Tube 2) and 6 (Tube 5). When they
came out of the incubator, we added 2 uL of the mix to 50 uL of competent cells. There
were four reactions total. They were then plated and left overnight but were unsuccessful,
Plate 1 may have had one colony, but it was hard to tell. We pelleted the leftovers and
resuspended them. The next day, we centrifuged them for 5 minutes. A pellet barley even
visible to the eye appeared. This could explain why the plates weren't successful: either
the math was incorrect or the concentration was not what we thought. Something had to have
been wrong, as 50 uL of competent cells were added to each aliquot, enough that should have
near guaranteed success. We plated the colonies and left them overnight. The plates were
extremely thin and weak when plating and broke in a lot of spots. Ultimately, the bacteria
was not still able to grow. We had to go through the Gibson assembly steps again. The competent
cells should have been good as they were straight from the NEB lab.
We started working in the summer with trying to get the TetR gene successfully inserted
into a plasmid with proper ribosome binding sites that will allow for more positive results
upon usage. Once we were able to successfully transfer the TetR gene with a 3A assembly method,
we mini-prepped her samples and sent them to M.S.U. for sequencing. We found no evidence of sufficient
base pairing, so unfortunately, all the work done over the Summer was unsuccessful. However, that meant
that there was more investigating to be done to improve the part. We started by doing PCR reactions
with prefix and suffix primers in one reaction and CUT 2 and CUT 4 primers in another. Gel electrophoresis
results showed that the DNA lengths were what we expected (approximately 1000 base pairs for the
prefix/suffix mix and 2000 base pairs for the CUT 2/CUT 4 mix), so we continued with a Gibson Assembly
and transformation, combining the K123002 with both cut 2/cut 4 and prefix/suffix. When beginning the
transformation, we decided to have one test tube as a control with no HiFi DNA assembly mix and one with
our HiFi DNA assembly mix. This allowed us to see a difference with our transformations and if there was
something wrong, both plates would show the same results. After incubation, the plate with the HiFi assembly
mix contained a reduced number of red colonies which is exactly what we wanted to and proved that there was
possibly some positive TetR colonies on the plate that assembled correctly.
We ran PCR reactions for 8 of the colonies without RFP, and then gel
electrophoresed them. That showed some colonies to be around 1000 base pairs
and others to be around 3000 base pairs.
We then did another round of PCR, combining 2 of the colonies each
with VF2 and TET sequence QR primers to confirm the presence of the
TetR gene. Additionally, liquid cultures were made of the same two
colonies and sent for sequencing, which yielded promising results.
Because of these results, we put our improved part in the parts
registry and renamed it K3737004.
The photo above is proof that K3737004 contains the stronger promoter, Part BBa_J23100.
The photo above is proof that K3737004 contains the human estrogen receptor element, part BBa_K3737003.
Above is the comparison of K3737004.
This shows that there was no significant similarity found between the two
sequences. Although this indicates that K3737004 does not contain
the RBS like we expected, we have improved upon K123002 in other
ways and will continue to work to include the RBS.
We made multiple liquid cultures to run procedures to confirm that we
succeeded in getting the K123002 strain into the plasmid. When comparing
K123002 to K3737004 via qPCR, we used the difference between CT values of
the strains with and without reverse transcriptase (RT) to calculate the
amount of RNA present. We used two clones of K3737004; both clones showed
lower CT values with RT than without. This proves that more DNA was made
from the RNA present in clones with RT compared to the clones without RT.
K123002 had very similar CT values. This means that converting the RNA
present did not make any more DNA, and there was no transcription of TetR
in the K123002 part. Because we used tet_seqR and tet_seqF primers, we know
that the TetR gene was present in K3737004 and not present in K123002.
The amplification plot made from the qPCR is a visual representation of our data.
We also made amplification plots to show the difference in amplification between
K123002 and K3737004.
The dissociation curve shows only one peak, proving that there is only
1 type of DNA in the qPCR samples. This proves that the tet_seqR and tet_seqF
were the only primers that were able to bind any genes in the samples.
This is promising because it indicates that clone 1 and clone 2 are the
same sequence. Based on this data, we have successfully improved upon K123002.
During our experiments, we ran into a few problems.
After our first PCR reaction, we found that our PCR
tubes had melted slightly under the temperatures we
had set for our reaction. Though the PCR tubes had
been melted, but the contents inside the tube were
still usable so we chose to move forward with the gel
electrophoresis. We ran into more issues with making
appropriate gels for the electrophoresis, with 2 failed
attempts taking up almost a full week of our time. Our
advisor helped us realize that during both gel preparation
attempts, we did not heat up the agar/LAB buffer mixture
long enough for the contents to dissolve properly, so we
solved our gel issue and were able to test our results.
Rainbow Trout Estrogen Receptor
We specifically used the Rainbow trout estrogen receptor because just like the
human estrogen receptor, it can bind tightly to estradiol. However, the RTER binds
to the DDT more efficiently than the Human estrogen receptor. Thus, making this a
start to the circuits we would be using. Why would I need to modify this though?
In the RTER, there were two locations in which ECOR1 is present. This strand
of base pairs can be present in the backbone but cannot be present in the strand
that we are replacing. It was considered illegal by iGEM. We started off this by
running the PCR reactions. A Gibson assembly reaction took place to bind the
two DNA strands that PCR reactions produced. A transformation was done to put
the DNA strands into cells to allow the cells to replicate the DNA. After about
a day, there were colonies that had formed. With this, we were able to run a
colony PCR to determine if we had the right number of base pairs in the DNA.
The first two showed total base pairs that were too low. However, the third lane
showed a band in between 2,000 and 2,500 base pairs. We were looking for between
1800 and 2200. This showed promise. I ran a mini prep and sent it in for sequencing.
When we got the sequences back, both ECOR1 sites had been removed giving this an
engineering success.
California Estrogen Receptor
We mainly decided to use the California Condor Estrogen receptor for the
same reason as the RTER. To start, we had to run the PCR reactions.
In one tube, we used the biobrick CC-er added with the forward primer
(CCER_Fix1_F) and the reverse primer (Cut4). In the other tube, same
biobrick but added the forward primer (VF2) and the reverse primer
(CCER_Fix1_R). After running a PCR, we got results for the first tube,
but not the second. So, we re-ran this PCR reaction at a higher temp.
at 67 degrees Celsius. This time, we got results for the second tube.
However, since there was smearing, we had to cut the DNA band that we wanted.
We then used this to extract the DNA. Using the tube 1 and the extracted DNA,
we ran a Gibson assembly and transformation. The next day, colonies had formed.
We created a liquid culture, mini prep and sent them off for sequencing.
When we got the sequences back, the ECOR1 site had been removed and
replaced which is engineering success.
