Experimental Timeline
As you can read in the introduction to our project, we used different proteins for different approaches. These proteins are in the following referred to by their PDB entries or abbreviations used in papers. For our optogenetic approaches, we used two different constructs: SOPP3-2ABH, consisting of 2ABH and the photosensitizer SOPP3 as well as VVD-CMI, consisting of the phosphate affine protein CMI and the fungal photoreceptor VVD. While working with the two constructs, it is important to work in the absence of light as the conformational changes in the proteins that define the optogenetic character may occur otherwise. Therefore, every eppendorf tube, falcon or column must be wrapped in aluminium foil while working with these constructs. Only in the final stage of the experiment, when the triggering of the conformational change is the goal, the proteins are exposed to light.
CW23 06/06/22-12/06/22
In our first week in the lab, we prepared phosphate solutions with different phosphate concentrations. These solutions were going to be used in our experiments later on. Our genes had not yet arrived, but we took the opportunity to let some of our lab members perform the Phosfinity Assay in order for us to be prepared for the coming weeks.
CW24 13/06/22-19/06/22
Because we were still waiting for our genes to arrive, we used the time to do more research. In particular, we focused on planning the experimental setup for our different approaches.
CW25 20/06/22-26/06/22
Since our genes still had not arrived yet, we continued with the research throughout the week.
CW26 27/06/22-03/07/22
Because our genes were to arrive soon, we prepared different media we would need for the transformation of the different genes and the production of cryocultures and competent cells. Therefore, we prepared LB liquid media and LB Agar plates, both with and without kanamycin, TFB2 medium for competent cells and glycerol stock solution. For the transformation and expression, we worked with Escherichia coli BL21 (DE3) gold as our host organism and pET-28a(+) as a vector. We prepared cryocultures of E. coli BL21 (DE3) gold containing the pET-28a(+) vector and cultures not containing the vector. Because we ordered the gene for 2ABH in isolated form as opposed to ordering it in a vector, we purified the pET-28a(+) vector we needed for the Gibson Assembly later. We decided to order 2ABH as an isolated gene in order for us to be able to perform a QuikChange later and thus engineering the protein in our favor.
CW27 04/07/22-10/07/22
We prepared TFB1 medium. After that, we generated competent cells from our E. coli BL21 (DE3) gold cryocultures, which did not contain the vector for the transformation with the vectors we ordered or produced with the Gibson Assembly.
CW28 11/07/22-17/07/22
At the beginning of the week, we prepared LB medium with kanamycin for agar plates and liquid medium. Since our genes had arrived, we transformed the competent cells with SOPP3-2ABH and VVD-CMI. Our first two tries for transformation did not work. Therefore, we changed the volume of the vector we used for the transformation. As a result, we were able to transform all the required genes. We also prepared SDS gels for the expression controls performed the following week.
In preparation for the Gibson Assembly, we amplified our isolated 2ABH gene and the purified pET-28a(+) vector via PCR. Through amplification, both, the isolated gene and the vector, have overlapping gene sequences, which were needed for the Gibson Assembly. To check if the PCR worked, we analyzed both constructs via agarose gel electrophoresis.
CW29 18/07/2022-24/07/2022
The week before we were able to generate different E. coli BL21 (DE3) gold clones carrying SOPP3-2ABH and VVD-CMI. For the use in the following experiments, we produced cryocultures for all of them. We also produced KPi buffer for storage of our cells and TB medium. We started with the expression of the proteins (VVD-CMI and SOPP3-2ABH), all expressions were conducted at 37 °C. To inoculate the expression cultures, we always used the cryocultures we generated. Because we worked with intracellular proteins, we could wash the cell pellet with KPi buffer after expression and store it at -20 °C until further use. Since we designed all our genes to have a $His_{6}-Tag$, we were able to use Ni-IDA columns for purification. We prepared Tris-HCl buffer, EDTA, Imidazole solution with different concentrations and NiSO4 solution, which are needed for the purification. First, we performed cell lysis via ultrasonication. Following the cell lysis, we separated the cell debris from the lysate supernatant via centrifugation. Afterwards, we were able to purify the proteins with Ni-IDA columns. To check if the expression and purification were successful, we ran a SDS-PAGE with samples taken at various time points. The SDS-PAGE from VVD-CMI showed that almost no protein was produced. SOPP3-2ABH showed more protein, but still not as much as we hoped for. This led us to change the expression temperature to 30 °C, at which we started another expression right away. The produced cell pellets were stored in the freezer to be analyzed the following week.
Since last week we generated 2ABH and pET-28a(+) genes with overlapping gene sequences, we were now able to perform the Gibson Assembly. Following the Gibson Assembly, we did a Check-PCR to analyze if the produced vector was carrying the 2ABH gene. Because this was the case, we were able to perform a transformation with the vector and competent E. coli BL21 (DE3) gold cells. We also sent a sample of the vector containing 2ABH to Microsynth for sequencing. The transformed cells were plated on LB agar plates containing kanamycin overnight in order to select positive clones.
CW30 25/07/2022-31/07/2022
At the beginning of the week, we got confirmation from the sequencing lab that 2ABH was in fact in the vector. Therefore, we prepared a cryoculture of the clone containing this gene and started the expression for the 2ABH protein as well. Before starting the expression, we had to prepare more TB medium. The expression was conducted at 30 °C and 37 °C overnight to assess the optimal conditions.
The week before, we already performed different expressions for VVD-CMI and SOPP3-2ABH at 30 °C, which we now had to analyze. Following the same procedure as the week before (cell disruption, centrifugation, purification) we controlled the different expressions via SDS-PAGE. Through evaluation of the different SDS-PAGEs we decided that 30 °C is the best expression temperature for SOPP3-2ABH and was therefore going to be used for this construct moving forward. VVD-CMI still showed low expression rates. To determine the exact protein yield, we decided to perform a Bradford Assay the next week. Another expression was started at 20 °C following some expert tips from our advisors.
CW31 01/08/2022-07/08/2022
Because we did the expression for 2ABH at 30 °C and 37 °C, we were able to compare the expression results through the same procedures as the week before. Building on that, we set 37 °C as the expression temperature for all following 2ABH expressions. The protein yield for 2ABH was already high, so we decided this should be the standard procedure moving forward. We performed another SDS-PAGE to check the expression and evaluate the optimal elution concentration of imidazole for the purification of the protein. Next to confirming the expression, we also decided to use two columns for the purification in the future since the sole column seemed to be heavily overloaded, which led to us losing a lot of protein.
Due to the fact that VVD-CMI showed only a small protein yield at 30 °C, we performed another expression at 20 °C last week. Unfortunately, the SDS-PAGE expression control led us to believe that the protein yield at 20 °C did not increase. In order to make a quantitative statement for all expressions and compare the exact yield at different temperatures, a Bradford Assay was performed this week. The assay confirmed high protein yields for 2ABH at 37 °C and SOPP3-2ABH at 30 °C as well as low protein yields for VVD-CMI at 30 °C and 20 °C. The goal for the following weeks was to improve the expression conditions for VVD-CMI.
Since we had now settled on expression temperatures for 2ABH and SOPP3-2ABH, both proteins were expressed again on a larger scale in order to maximize the amount of protein we could recover. The culture media were centrifuged after incubation, and the pellets were stored at -20 °C until further use.
CW32 08/08/2022-14/08/2022
After the purification of 2ABH and SOPP3-2ABH proteins produced last week, we were now ready to test for phosphate binding capacity, and in the case of SOPP3-2ABH releasing capacity under the influence of blue light. Of course, we had to confirm a successful expression and purification, so we performed an SDS-PAGE again. After purification, we performed a buffer exchange using an Amicon column. This allowed us to dissolve the protein in HEPES buffer instead of the imidazole, in which it was still dissolved in, as well as to reduce the total volume of the buffer to two to four milliliters. The proteins were then stored on ice at 4 °C overnight until immobilization the next day.
For the immobilization, we were able to use the $His_{6}$-Tag, which was also used for purification. As an immobilization matrix we used EziG beads. Since there are three different types of EziG beads, we had to test which type is best suited for our proteins. The immobilization was performed on all three types in the same way. By determining the concentration of proteins via Bradford Assay before and after bead binding, it was possible to determine which bead type bound the most protein. In our case, this was the opal EziG bead type for both 2ABH and SOPP3-2ABH, so going forward we used the opal beads only.
To determine the phosphate binding activity of the immobilized proteins, we exposed them to a phosphate solution with known phosphate concentration. After 30 minutes of incubation, while shaking, we took a sample from the supernatant and photometrically measured the phosphate concentration with the Phosfinity Assay. The results showed that both proteins were still able to bind phosphate while being immobilized on EziG beads. After this initial success, we now exposed the SOPP3-2ABH protein construct with bound phosphate to blue light to test for the release of the phosphate molecules. We took samples at different points in time and measured the phosphate concentration.
We also ordered the primers designed to incorporate specific mutations in our 2ABH gene via QuikChange.
CW33 15/08/2022-21/08/2022
To replicate our results, we repeated the experiments from last week for 2ABH and SOPP3-2ABH. Because in the weeks before we noticed that the measurement of the protein concentration via Bradford Assay showed some fluctuation, we decided to test two new methods. First, we measured the protein concentration with NanoDrop, which unfortunately showed even higher fluctuations. We then tested the BCA Assay, which proved to be the most stable method. As a result, we only used the BCA method for determining protein concentration going forward. After establishing that, we repeated the experiments from last week. We tested for the phosphate binding capacity of both 2ABH and SOPP3-2ABH and for the phosphate releasing capacity of SOPP3-2ABH.
During the week we also generated new 2ABH and SOPP3-2ABH pellets, which means we expressed both proteins as described above and stored the washed pellets at -20 °C for later use. For our VVD-CMI approach, we did not have the right expression conditions yet. As a result, this week we started three different approaches to get better results. We varied the media we used (TB medium, LB medium and autoinduction medium) and carried out the expression at 18 °C for 48 hours. At the end of the week, the resulting pellets were washed and stored at -20 °C, until the expression control was performed the following week.
We also produced a new KPi buffer this week.
CW34 22/08/2022-28/08/2022
The week before, we tried to express VVD-CMI in different media and conditions in order to get a higher protein yield. For expression control and comparison, we used the SDS-PAGE, which showed that the most protein was produced in the autoinduction medium. Therefore, we expressed VVD-CMI under these conditions in a larger volume in order to produce high quantities of protein. The produced pellets were washed and stored at -20 °C. During the week, we also tested the immobilized SOPP3-2ABH for the binding of phosphate and phosphate release after exposure to blue light. We used the same methods as the weeks before.
We also attempted to mutate 2ABH with the primers we ordered by performing a QuikChange. The agarose gel electrophoresis we conducted to verify the success of the QuikChange showed that it failed though. Because of the limited time we had left in the lab and considering the required effort to continue working on this approach, we decided to discard the optimization of 2ABH and instead focus on the optogenetic switches. This might still be an interesting point of evaluation for future iGEM teams or research groups.
CW35 29/08/2022-04/09/2022
The testing for SOPP3-2ABH was repeated twice this week, using the pellets we produced the week before. As before, we disrupted the cells, purified the protein, exchanged the buffer and immobilized the protein before performing the illumination procedure itself. The testing methods also remained unchanged. To ensure that all our expressions worked, we conducted a collective SDS-PAGE with samples taken before, while and after purifying the proteins.
We also carried out two more expressions, one for SOPP3-2ABH and VVD-CMI each, under the previously defined conditions. The pellets were washed and stored at -20 °C until further use.
Furthermore, we produced a new TB medium, imidazole solution and KPi buffer.
CW36 05/09/2022-11/09/2022
Two pellets, one of 2ABH and one of SOPP3-2ABH were disrupted and the proteins were purified. After purification, KPi buffer was added and the protein solutions were stored overnight on ice at 4 °C. In the purification process, there were samples taken to verify the presence of the respective protein via SDS-PAGE as it was done the week before.
After that, the proteins were immobilized and tested for phosphate binding capacity. In the case of SOPP3-2ABH, the phosphate releasing capacity was determined using previously defined methods. The success of the immobilization was tested with the BCA Assay and to quantify the bound and released amount of phosphate, we used the Phosfinity Assay.
We also started with the production of polyphosphate in yeast cells. Therefore, we set up an overnight culture in SD medium and used that to inoculate a bigger culture, also in SD medium, the next day. After one day of incubation, we transferred the cells into a starvation medium. After further incubation time, we washed the cells with $ddH_{2}O$ and stored them at 4 °C. The next day, the cells were put in a feeding medium, which we produced beforehand. After incubation, the cells were washed with $ddH_{2}O$. We then weighed four cell samples of 25 mg each and stored them with the rest of the cell mass at -20 °C until further use.
CW37 12/09/2022-18/09/2022
In this week, we purified and immobilized two VVD-CMI pellets, taking samples for SDS-PAGE and BCA Assays as usual. After that, we tested for phosphate binding capacity in the same manner as we did for all other approaches. The phosphate releasing capacity was tested by exposing VVD-CMI to blue light in a phosphate solution and keeping it in darkness with bound phosphate for 5 hours. We were taking samples for a Phosfinity Assay every 30 minutes.
For the assessment of the amount of produced polyphosphate, we extracted the polyphosphate from the yeast cells with chloroform and phenol. These samples were used to perform a Phosfinity Assay and thus determine the mass of produced polyphosphate. The samples from earlier this week were analyzed using the assay as well.
After this week, we decided to stop the lab work and focus on lab cleaning and composing our wiki.
