Assembly of the Circuit
Matthew Hanson, Sebastian Nolte, Rachel Kostrzewa
Background
The creation of the DDT biosensor comes with three different parts that are assembled together. These three parts are the rainbow trout estrogen receptor(rtER), tetracycline repressor (TET), and the red fluorescent protein (RFP). The rtER acts as a binding site for estrogen. Why use this for detection of DDT? DDT has a similar chemical structure to that of estrogen as the aromatic rings line up with one another. Because of this, it allows for DDT and its derivatives to bind to the site. The rtER acts as the first part of the circuit for the biosensor. This is followed by the TET gene, then the RFP gene. In this circuit, when DDT is not present, the TETgene represses RFP which means the cell will not turn red as RFP would cause this to happen. However, when the circuit is exposed to DDT, the rtER activates. When rtER activates, it represses the TET gene. Because of this, TET cannot repress RFP anymore so the cell will turn red.
Assembly
The assembly of the had multiple different aspects and routes that could have been taken. The first way the circuit was attempted to be assembled was through the sequence of rtER-TET-RFP. For this, a PCR reaction was set up for assembling the genes with the primers needed to react. These three pieces went through the designed PCR protocol. rtER was assembled with the forward primer VF3 and reverse primer RT-R1. The TET was assembled using the forward primer TET-F1 and the reverse primer TET-R1. Finally, the RFP primer was assembled using the forward from RFP-F2 and the reverse primer VR3. After assembly of these genes, a gel electrophoresis was used to determine if the desired base pairs were found. Below are two Gels. The first is the gel with the TET and RFP which had the correct number of base pairs (Column 3 and 5). The second gel was three rtER reactions which resulted in the correct base pairs.
After the desired base pairs were achieved, the pieces were now used in a Gibson Assembly and a Transformation. After plating, the assembly fell short. There were no colonies present. Each time, re-running the reaction, the same result would occur. No colonies would be present. This causes for two theories. The first being that the DNA strand may be too long for the cell. Thus, it may put too much burden on the cell making it not be able to replicate the DNA. The second theory being that it was toxic to the cell. However, this was very unlikely because each piece had been assembled in a cell prior to the assembly of the circuit.
The other possible way to create this circuit was the circuit did not have to be assembled all together in one plasmid. It could be assembled in multiple different plasmids. Then, all transformed into one cell. Because of the possibility that the circuit was a burden for the cell to replicate, it was decided that the TET and RFP genes from the previous experiment would be assembled together and the rtER would be assembled into a different backbone. For this, the backbones had to be produced. The backbones that we used during this assembly each had a different antibiotic resistance gene on them: Chloramphenicol, Ampicillin, Kanamycin, and Tetracycline. Each one of these is important when growing the cells after transformation because each one of our plates contain one of these antibodies. For the cell to grow, the cell would have to have the antibiotic resistance gene that matches the antibody on the plate. This allows for a controlled environment so no different cells will grow on them. To make these backbones to bind to the specific genes, the Cut 4 and Cut 2 primers were used. The Cut 2 primer acts as the forward primer and the Cut 4 acts as the reverse primer. For the rest of this reaction, follow the designed PCR protocol. After these backbones were synthesized, a Gibson assembly was run using the four backbones. Both the rtER and the TET plus RFP were combined separately into each of the back bones totaling eight Gibson Assemblies. Each one of these were then transformed and plated onto the corresponding plates. After they were incubated, the rtER was seen to be present on all the plates. However, on three of the plates that were supposed to have TET plus RFP, there were no colonies. On the Kanamycin plate, there were many colonies as well as it was very streaky. Based on the concentration of Kanamycin that we used, the cells should not grow that rapidly. Because of this, it was deemed that there was something wrong with the Kanamycin and the results were inconclusive. Over the whole season, there has been some success but not much when it came to assembling the circuit.
Transposons
Transposons were one of the main issues we ran into over the season. When running PCR reactions using the TET gene, the TET should be at 1,000 base pairs. However, randomly in the middle of the summer, the TET gene started to record base pairs of 2,000. How did this happen? We troubleshooted by changing the annealing temperature of the PCR reaction. We changed the concentrations of the primers and DNA. We also changed the cycles ran just in case the DNA was duplicating itself twice. None of these seemed to work. We sent in some of the DNA to get sequenced. Once the sequences had returned, we compared them to other DNA data banks. From this, we saw that the DNA matched to multiple different transposons. A transposon is a jumping gene. At specific base pairs, a transposon will bind to the DNA. At this point, it will cut the DNA until it hits a codon indicating for it to stop. Once it hits this, it stops cutting. There is now an open space present in the DNA. The transposon will then bind into the open spot thus causing a mutation in the DNA. This was seen to be present in the TET gene. This issue caused us to be set back three weeks because we had to turn our sites to the origin of the problem.
Next Steps
As of right now, there is a plate with colonies of supposably all three genes. We will not know until we get the sequence back. Assuming that the sequences come back with the exact bases that we want, then the circuit would be complete. From there, we will test to see if the circuit works. We would transform the DNA into the E Coli cells which would then be plated. On one plate, we would put estrogen, and on the other, no estrogen would be present. After incubating the cells, the colonies on the plate with the estrogen should turn red, and the other colonies on the other plate should stay a beige color. Assuming this does happen, we can then go to test it even further with testing the concentrations of estrogen in which the biosensor works. This would show the limits of where our biosensor can detect DDT.
Cell Free Protein Synthesis (CFPS)
CFPS is the process where proteins can be synthesized and expressed outside of the cell with less constrictions. The idea we had for using this was using it for our circuit. Because we could not get the circuit to come together as one, we think that we can get the genes to express individually which would allow for our circuit to run in full without having to be assembled together. One main benefit with us doing this is we can choose the concentration of genes that we want to use. We are also using this process to bypass gibson assembly and transformation. Any issues that can occur during Gibson assembly and Transformations can be cut out of the equation entirely.
Below, is how we made the CFPS solution:
To make our LoFT solution we made a large culture of Escherichia coli BL21(DE3) codon plus (RIL) in 1 L of fresh LB medium in a concentration range of .1% to 1% while shaking at 120 rpm at 37°C. After 1 hour, IPTG was used at a concentration of .1 mM to start the expression of T7 RNA encoded in DE3 under the promoter of lacUV5. The collected cells via centrifugation were at OD600 = 1.0–2.0 then suspended in 20 ml of 400 mM of sucrose. At four separate points 50 μL of 20 mg/mL lysozyme was dissolved in 400 mM sucrose and then added to the cell's suspension. This gives a final concentration of the lysozyme of .2 mg/mL. Tubes were then shaken by inversion and incubated on ice for 30 minutes. Cells were then washed with 20 ml of 400 mM child sucrose twice. Cells were quickly resuspended with a paintbrush. The washed cells were centrifuged and collected then dissolved in DDW. Concentration varied but usually 1 mL per g of wet cell paste. Cells were then transferred to 1.7 mL tubes and then frozen at -80°C for 1 hour. After thawing and being centrifuged at 25,000 x g for 1 hour, the supernatants from the cell were collected as LoFT extracts and stored at -30°C.
Tests
In order to determine how the CFPS works and how effectively, two separate experiments were set up. The first was testing to see how specific this reaction had to be to the correct concentrations. The LoFT solution was diluted to a concentration of 2 mg/mL. The other solution containing the enzymes, amino acids, and other essentials (seen in the table below) was diluted to four different concentrations: 1/40, 1/50, 1/75, and 1/100. 45 uL of this solution was combined with 50 uL of the LoFT solution. This solution was then tested using 5 uL of RFP. The solution was then set in a 30 Celsius incubator which sat overnight. After letting it sit for 18 hours, the results were recorded. What was expected was that the solution would turn red as the RFP gene would be expressed. However, none of the solutions turned red. Therefore, it can be said that Cell Free Protein Synthesis has to be near to the exact concentrations needed for the solution to work. Because of this, new solutions had to be produced. Using the table below, the new solution of the amino acids and enzymes were produced at higher concentrations. This would allow for us to dilute these in a solution to their exact concentrations. The second experiment of testing the CFPS is still a work in progress.
| CURRENT CONCENTRATION (M) | DESIRED CONCENTRATION (M) | uL | |
|---|---|---|---|
| NAD+ | 0.5 | 0.00033 | 0.66 |
| ATP | 0.5 | 0.0015 | 3 |
| CTP | 0.05 | 0.0009 | 18 |
| GTP | 0.05 | 0.0015 | 30 |
| UTP | 0.05 | 0.0009 | 18 |
| E | 0.005 | 0.0005 | 100 |
| W | 0.05 | 0.0005 | 10 |
| R | 0.5 | 0.0005 | 1 |
| C | 0.5 | 0.0005 | 1 |
| S | 0.5 | 0.0005 | 1 |
| G | 0.5 | 0.0005 | 1 |
| K | 0.5 | 0.0005 | 1 |
| P | 0.5 | 0.0005 | 1 |
| A | 0.5 | 0.0005 | 1 |
| HEP | 1 | 0.05 | 50 |
| PEG | 0.2 | 0.02 | 100 |
| MAL | 1 | 0.012 | 12 |
| MgACT | 1 | 0.014 | 14 |
| SPERM | 0.003 | 0.001 | 333.333333 |
| FA | 0.068 | 0.000068 | 1 |
| CAMP | 0.146 | 0.00075 | 5.1369863 |
| TRNA | 0.2 | 0.0002 | 1 |
| KE | 0.5 | 0.09 | 180 |
| Y | 0.0025 | 0.0005 | 200 |
| V | 0.25 | 0.0005 | 2 |
| F | 0.1 | 0.0005 | 5 |
| T | 0.25 | 0.0005 | 2 |
| M | 0.1 | 0.0005 | 5 |
| H | 0.1 | 0.0005 | 5 |
| I | 0.1 | 0.0005 | 5 |
| L | 0.1 | 0.0005 | 5 |
| N | 0.091 | 0.0005 | 5.49450549 |
| Q | 0.167 | 0.0005 | 2.99401198 |
| IPTG | 0.1 | 0.001 | 10 |
| COA | 0.013 | 0.00026 | 20 |
| 3PGA | 0.1075 | 0.036 | 334.883721 |
| D | 0.1 | 0.0005 | 5 |
Next Steps
With the solution complete we would be able to test our circuit and other factors. We would first test the solution with RFP and the solution would turn red if our CFPS solution was made correctly. Assuming this occurred, we would then test our circuit. In this test, we will create the same solution as before. But this time, RTER, TET, and RFP would all be added to the solution. The concentration of these genes would be the same so one expression does not outweigh the others. After this solution is incubated, we would expect to see a clear color present. If this occurs, then we would then add estrogen to the solution. This solution would then be incubated overnight. If the expression worked, the solution would turn red showing the circuit is complete.