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.
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.
The above results were then sent to MSU for Sanger Sequencing
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.