Go to Previous Work & This Year's Plans
Go to New Composite Part: araC Inverter
Go to Engineering Success: Full Circuit Using TetR
The Biosensor Circuit
Background
The DDT biosensor is assembled using three different parts: rainbow trout estrogen receptor (rtER) (K3737001), tetracycline repressor (TetR) (K3737004), and red fluorescent protein (RFP) (I13521). The rtER acts as a binding site for estrogen. This is used for the receptor for DDT as DDT has a similar chemical structure to that of estrogen. The aromatic rings line up allowing for DDT and its derivatives to bind to the site. In the circuit, rtER is followed by the TetR gene and then the RFP gene. RFP causes the cells to glow red. When DDT is not present the TetR gene represses RFP resulting in no red color in the cells. But when the circuit is exposed to DDT, rtER activates and represses the TetR gene. This repression of TetR results in the expression of RFP causing the cells to turn red and can be seen in Figure 1.
Figure 1
Previous Work & This Year's Plans
Go to TopThe basis of our biosensor is to use E. coli to produce red fluorescent protein (RFP) in the presence of DDT. In the 2021 cycle, the rainbow trout estrogen receptor was chosen as it binds tightly to estrogen like molecules. Activation of the estrogen receptor inhibits the TetR protein which results in RFP production. Estrogen receptors are activated by DDT and its derivatives acting as the ligand.
In the 2022 cycle, the rtER, TetR, and RFP genes were assembled together on a single backbone. These plasmids were further transformed into E. coli such that in the presence of DDT, RFP will be expressed. By the end of the cycle, rtER expression was successful, but TetR and RFP expressions failed.
This year our goal is to continue troubleshooting the circuit to see how we can get the full circuit to function and what the minimum concentration of DDT our biosensor can detect. We have considered other possible means to effectively complete the circuit in E. coli to get RFP expression. So far, LuxR and C2 alternatives for the primary circuit have been troubleshooted to see if they could be possible circuit candidates. But, the transformations were not successful. These were intended to be back up options in case there were further issues. We have produced a new inverter (araC) and fixed the TetR inverter. We are currently testing different concentrations of araC and TetR to determine the amount of fluorescence expressed by each.
New Composite Part: araC Inverter
Go to TopWhile reflecting on our engineering obstacles from our previous cycle with attempting to use the TetR inverter, it was decided with a substantial literature review, that the TetR inverter would be better replaced with the araC inverter. Similar to the TetR inverter, the araC inverter contains three pieces. The first piece is the rtER promoter (J23100).This promoter contains the rtER_E (rtER response element) (K4732000). This response element acts by binding to rtER in the presence of DDT which blocks transcription of the araC repressor. The next two pieces come together: the araC repressor and the araC promoter (Q04800). Without the presence of DDT, the araC repressor blocks the araC promoter from being expressed.
When DDT is present, the rtER binds to the DDT which in turn activates the response element causing for araC repressor not to be transcribed. Due to this, the araC promoter is not blocked allowing for it to be expressed allowing for the transcription of RFP.These pieces were created and assembled together using PCR, gibson assembly, and transformation. After the colonies grew, we began a series of tests to determine if the araC worked. Initially, we created plates with different concentrations of 5% arabinose, a sugar that blocks the araC repressor. If the araC repressor is working, the arabinose is expected to repress the repressor which would lead to the expression of the RFP. Our experiment consisted of six plates, which were made with different volumes of 5% arabinose (200 μL, 100 μL, 50 μL, 25 μL, 15 μL, and 7 μL).
Figure 2
As seen in Figure 2, the arabinose was detected at all the volumes down to 7 μL of 5% arabinose. This gave a concentration of 93 μM arabinose detection. This result proves that the araC inverter was produced within our experiment and works. Next, we wanted to further verify our data, results, and validity of the araC inverter by testing it in estradiol (E2). However, trials in E2 were not a success, due to too much of the araC repressor being transcribed. Because of this, E2 was not able to repress enough araC repressors to allow for the expression of RFP. To workshop this issue, we tested the lowest concentration of arabinose needed to repress enough araC respressors to express RFP. Figure 3 below shows the lowest concentrations of arabinose needed for RFP expression:
Figure 3
From the results graphed in Figure 3, we were able to detect RFP down to 16.0 μM of arabinose. This result is important because using a lower concentration of arabinose will allow for RFP expression. However, it will still be a faint red meaning that araC is still repressing the RFP from its max fluorescence.
From here, we decided to test the full circuit, but this time adding the 66.6 μM of arabinose, Isopropyl-ß-D-thiogalactopyranoside, known as IPTG, as well as different concentrations of E2 to the liquid culture. Four liquid cultures were produced. Each of the four liquid cultures contained 66.6 μM of arabinose and 25 μL of IPTG. The only thing that changed was the amount of E2 we added to each of the liquid cultures. One of the four liquid cultures contained no E2 to act as our control for this procedure.
Figure 4
Looking at Figure 4, the control group produced the smallest amount of red fluorescence. At 30 μM of E2 compared to the control, RFP expression increased by 14%. At 60 μM of E2 compared to the control, RFP expression increased by 36%. At 120 μM E2 compared to the control, RFP expression increased by 46%. These results showing an increase in RFP expression support that the araC inverter works when genetically integrated into the full circuit.
Engineering Success: Full Circuit Using TetR
Go to TopIn the previous year, the full circuit of the biosensor for DDT was completed. However, it never responded to E2 giving no expression of RFP. Over the summer, pieces were sent in for sequencing. It was determined that half of RFP was missing. The main piece missing was the tetracycline promoter. To troubleshoot this, the team re-ran PCR with a higher annealing temperature. This produced the piece that we needed. However, after sending and receiving the sequences for the circuit again, it was determined that there was a point mutation at the start codon causing RFP not to be expressed. After a few more PCRs and assemblies, the circuit was fully assembled. When looking at the bacteria with the circuit, the bacteria expressed a faint red color, shown in Figure 5.
Figure 5
This indicates that RFP is being expressed by the circuit. However, the TetR repressor is making it so the RFP is not expressing the maximum red fluorescence. These bacteria were suspended in four separate liquid cultures with different substituents combined in the LB. The first liquid culture contained DMSO which would act as the control group. The second liquid culture contained aTc which would repress TetR causing RFP to be expressed. The third contained 10 μM E2 which would bind to the rtER causing for the TetR repressor to be repressed leaving RFP to be expressed. The last liquid culture contained a combination of 10 μM E2 and aTc. The results of the plate reader are shown below in Figure 6.
Figure 6
When DMSO was compared to aTc, the liquid culture with aTc increased in expression by 20%. When DMSO was compared to E2, the E2 culture increased in expression by 22%. Lastly, DMSO was compared to the combination of E2/aTc which showed an increased expression by 34%.This indicated that we have successfully produced the circuit and have determined that RFP can be expressed in E2. We did not stop here.We wanted to test the lowest concentration of E2 that would show the expression of RFP. Again, four liquid cultures were produced with different concentrations of E2 (40 μM, 20 μM, 10 μM, and 6.7 μM). The results of fluorescence are shown below in Figure 7.
Figure 7
Figure 8 below, shows the fluorescence of RFP was seen in concentrations of E2 at 6.7 μM and above. However, the graph shows randomness to it. When looking at the 10 μM E2 compared to 6.7 μM E2, the expectation should be that 10 μM E2 should cause more expression of RFP. However, 6.7 μM E2 caused a higher expression than 10 μM E2. Though this is the case, it can be determined that the circuit is able to express RFP all the way down to 6.7 μM of E2.
Figure 8
Figure 9 below, shows the TetR circuit from left to right at the following measurements: 40 μM E2, 20 μM E2, and 6.7 μM E2.
Figure 9