Notebook and Experiments

Our first day in the lab for the 2023 UAlberta iGEM team was Monday, May 8th. Before lab work commenced, we received eight pBAD plasmids containing eight different fluorescent proteins; pBAD+eYFP, pBAD+mClover3, pBAD+mTurquoise2, pBAD+mScarlet, pBAD+mCitrine, pBAD+sfGFP, pBAD+mNeonGreen, and pBAD+mTopaz. We started the week by doing lab preparation work with the ultimate goal to transform the eight pBAD plasmids into Escherichia coli DH5ɑ, one of the most utilized lab cloning strains due to its high-efficiency transformations, good plasmid quality prepared from minipreps, and high insert stability(1) as well as E. coli Rosetta-gami (DE3), a protein expression stain optimized for enhanced disulfide bond formation and expression of eukaryotic proteins containing rare codons(2). We were able to complete the transformations, allowing us to visually observe the relative fluorescence of each protein when expressed by Rosetta-Gami on plates. The cells were plated on LB+Amp+2% Arabinose agar plates to induce protein production in the Rosetta-Gami cells, though the results from the Rosetta-Gami transformations were subpar in comparison to DH5ɑ . We also ordered primers to amplify the eight fluorescent proteins for cloning in future weeks. The goal of week 1 was to provide data to our tech team and begin determining which fluorescent protein to incorporate into our planned sensor construct.

The second week in the lab involved repeating the pBAD-fluorescent protein transformation in Rosetta-Gami to obtain better results; we also modified our plating strategy for transformations. We inoculated the transformed DH5ɑ plated colonies from Week 1 into liquid cultures which were subsequently turned into glycerol stocks to maintain the pBAD plasmids containing eight different fluorescent proteins. We also obtained the pET-22b(+) expression vector in a glycerol stock, which we inoculated into a liquid culture, and isolated using the QIAprep Spin Miniprep Kit. The pET-22b(+) vector is optimized for the cloning and expression of recombinant proteins in E. coli. Genes cloned into the pET-22b(+) vector are under the control of bacteriophage T7 transcription (a strong T7 promoter); expression is induced by providing a source of T7 RNA polymerase in the host cell(3). We subsequently double-digested the isolated pET-22b(+) with HindIII-HF and NdeI, and verified the digestion went to completion using agarose gel electrophoresis. We closed the week by performing a gel extraction of the double-digested pET-22b(+) to purify and isolate the backbone for our planned clonings to take place in Week 3. The goal of week 2 was to ensure long-term storage of the fluorescent protein genes expressed in week 1, and prepare the pET-22b(+) expression vector for our future planned cloning and expression experiments.

The primers we ordered in Week 1 to amplify the fluorescent protein genes out of the plasmids we received arrived and thus we began our first attempt at Gibson assembly. We started Week 3 by amplifying the fluorescent protein genes out of the plasmid vector in which they were contained using Q5 High-Fidelity DNA Polymerase. Our first PCR reaction had poor results but on our second attempt, all fluorescent protein inserts with the exception ot mNeonGreen were amplified successfully. We consistently had issues amplifying mNeonGreen that we had to workshop until we realized that the primers had been designed for a different template plasmid, donated separately of the original 8 plasmids we received. Once this problem was recognized, we were able to rectify the issue and successfully amplify mNeonGreen. We PCR purified eYFP, mClover 3, sfGFP, mCitrine, and mTurquoise 2 as no non-specific amplification was observed when the PCR samples were run on a gel. We performed a gel extraction on the mScarlet, mNeonGreen, and mTopaz amplifications as they exhibited non-specific amplifications. We subsequently quantified the DNA of the PCR purified samples using Agarose Gel Electrophoresis, and set-up our first Gibson assembly reactions using eYFP, mClover 3, sfGFP, mCitrine, & mTurquoise and the digested pET-22b(+) vector from Week 2. Unfortunately, these reactions proved unsuccessful and we returned to the drawing board. We concluded Week 3 by preparing materials and protocols to re-attempt the fluorescent protein pET-22b(+) Gibson Assembly in Week 4 and focused on troubleshooting the obstacles we encountered. Week 3 resulted in being an invaluable learning opportunity for our team as we ventured into more difficult techniques such as PCR and Gibson Assemblies.

This week we focused on rectifying the issues we encountered with our Gibson assembly reactions in Week 3. We began the week by repreparing our pET-22b(+) vector as its previous preparation was identified as one the potential sources for our unsuccessful results. We extracted the plasmid and performed an overnight restriction enzyme digestion with HindIII-HF and NdeI and were extremely prudent when interpreting the agarose gel electrophoresis results and performed a gel extraction to ensure the backbone was properly digested and able to accept inserts. We proceeded to set up our revised Gibson assembly reactions using the digested and gel purified pET-22b(+) vector as well as the eYFP, sfGFP, mScarlet, and mNeonGreen inserts from Week 3. We elected to only set-up four reactions to conserve the few aliquots of Gibson master mix we had been donated and because we had yet to attempt a reaction using the mNeonGreen and mScarlet inserts. We subsequently transformed the four Gibson reactions into DH5ɑ, performed a colony PCR to verify the inserts were present in the cells, extracted the positive colonies plasmids, and sent the extracted plasmids for Sanger sequencing to verify the success of our cloning. Preliminary agarose gel electrophoresis of the Gibson reaction suggested that our cloning was successful but this could only be confidently concluded once the sequencing results were returned. In addition to continuing with our cloning experiments, we performed a verification test to verify whether a buffer-based lysis method was able to extract and purify the fluorescent proteins when protein production was induced in E. coli Rosetta-Gami. The supernatant obtained from this lysis method would theoretically contain any soluble proteins within the cell. We utilized a fluorescence microplate reader to analyze the relative fluorescence of the purified protein samples and obtained results that suggested the proteins were being expressed properly by the bacteria and were detectable with the methods we employed. Week 4 allowed us to resolve the issues we encountered with our Gibson assembly reactions in Week 3 while we continued to move towards finishing our first experiment characterizing fluorescent proteins in the pET-22b(+) vs pBAD expression systems and also verify that the fluorescent proteins we were utilizing were being properly expressed by the bacteria. These results offered insight into the future plans for our project in relation to our sensory construct that would ultimately be designed using the fluorescent proteins we chose from our initial cloning and expression experiments.

With the completion of our first cloning experiment using Gibson Assembly to combine pET-22b(+) with four different fluorescent proteins, we received the sequencing data to confirm the success of the cloning and thus moved onto the next phase of our project. We attempted to transform and express the pET-22b(+) fluorescent protein constructs in Rosetta-Gami induced on plates containing IPTG but observed inconsistent results. After receiving input from the technology team and analyzing the expression of the fluorescent proteins, we ultimately decided to use mScarlet in the design of our sensory construct. Our dry lab got to work designing what ultimately came to be known as the Q-sensor construct, an interim design on the path to creating Fungalescence. The dry lab also modeled and produced the antigen fragment we would express and purify to perform the nanobody library screening to identify a binder for the antigen fragment. For more information on the development and modeling of the antigen and Q-Sensor constructs, please refer to the dry lab page of our wiki. With this in mind, we entered an interim of expectancy as the constructs were designed, ordered, and shipped. During this period, wet lab activities were minimal but we continued to have weekly meetings where we developed the cloning strategy and the workflow for the rest of our project and focused our efforts in other areas, principally human practices. Near the end of Week 7 we realized that there had been a miscommunication with IDT and our antigen and Q-sensor constructs would be arriving in a pUC vector instead of a pIDTSmart vector. This led us to redesign our Gibson Assembly cloning strategy of these two constructs in the pET-22b(+) vector.

After we realized there had been errors in the ordering of our constructs from IDT, we were unable to employ the original strategy we developed where we would utilize restriction cloning to insert our Q-Sensor and Antigen construct into the pIDTSmart vector. The pUC plasmids the constructs were provided in lacked a terminator thus we had to introduce one into our reading frame. To resolve the issues, we decided to redesign our cloning strategy to generate a linearized pET-22b(+) backbone with overhangs that would be compatible with the overhangs generated in a PCR reaction containing our Antigen and Q-Sensor constructs, respectively. From the pET-22b(+) backbone, we would include the T1 terminator and from the pUC vector containing the constructs, we would include the T7 promoter and insert sequences. This week we were able to generate PCR fragments that corresponded with our linearized backbone as well as with the antigen, however the PCR to generate the Q-Sensor insert was unsuccessful. In light of this, we re-performed the PCR reactions for the backbones, the antigen insert and the Q-Sensor to generate more DNA product but the opposite issue manifested and only the PCR to generate the Q-Sensor worked, no antigen fragment was detected. With these inconsistent outcomes of our PCR reactions, we began brainstorming ways to optimize the PCRs. We decided that one method we could attempt would be to linearize the pET-22b(+) template DNA to make it more accessible to the primers and the Q5 polymerase.

After struggling with our PCR linearizations for the majority of Week 10, we were able to successfully generate a linearized backbone compatible with a Q-sensor insert after modifying the reaction conditions (using an increased concentration of dNTPs, diluting template to 1 ng/µL). Despite this success, the pET-22b(+) backbone compatible with the antigen fragment was still giving us trouble. We decided to use a lower fidelity polymerase, DreamTaq, to help offset costs and help us amplify our large fragment which proved to be the correct choice as we were able to generate our antigen-compatible backbone. The PCR reaction for generating the Q-sensor insert was also redone and we were able to successfully generate our Q-Sensor insert. We then proceeded with performing a Gibson assembly of our antigen fragment into the compatible linearized pET-22b(+) backbone. After the Gibson assembly reaction was completed, we first analyzed the reaction on an agarose gel to see if we could tell a difference between the template pET222b(+) plasmid and our cloned construct containing the antigen. We then transformed our gibson assembly into DH5a competent cells and were successful in obtaining colonies.These colonies were then subjected to colony PCR where successful transformants were then identified. At the same time, we purified our Q-sensor insert and prepared to perform another Gibson assembly. Using the same method to assemble the pET-22b(+) and the antigen construct, we estimated the amount of DNA needed for the pET-22b(+) + Q-Sensor reaction from a gel, performed the reaction, analyzed part it on a gel, and transformed the remainder of the reaction into DH5a competent cells.

The transformations of DH5ɑ with the Q-Sensor Gibson assemblies resulted in a few colonies we were able to Colony PCR using the primers that generated our insert. We then prepared overnight cultures of the successful transformants from both the antigen and Q-sensor assembly to prepare for minipreps We then sent the minipreps from the positive colonies of both the pET-22b(+)-Antigen and pET-22b(+)-Q-Sensor construct for sequencing and confirmed that our Gibson assemblies were successful. Moving forward, we then sought to induce high levels of protein expression so we could subsequently purify our antigen for the nanobody selection protocol. We transformed our minipreped DNA of the ligated constructs into Rosetta-Gami competent cells and allowed them to grow overnight. The results of the pET-Q-Sensor transformation were uplifting as the transformed colonies appeared purple in colour, potentially due to the presence of the ShadowR protein in the construct being expressed. This week marked a turning point as we had successfully assembled the pET-22b(+)-Q-Sensor construct and the pET-22b(+) -Antigen construct we had ordered in Week 6.

With the success of our Gibson Assemblies of the pET-22b(+) vector with the Antigen construct and the Q-Sensor construct in Week 12, the next step in our project would be to characterize and purify both of the proteins. One of the key steps in protein identification is the use of SDS-PAGE gel electrophoresis which separates proteins based on their molecular weight. This week in preparation for the protein production and purification assays ahead, we worked on pouring SDS-PAGE gels for future use.

We prepared to hopefully purify our Antigen and Q-Sensor constructs from our transformed Rosetta-Gami cells by preparing overnight cultures and the buffers we needed. We induced protein expression using 0.1M IPTG in 100mL of Rosetta-Gami culture. We then split the induced culture to test two different methods for protein purification, Buffer/Bead based lysis and French press lysis. After we had obtained the cell lysate, we then used preloaded Ni-NTA columns to purify our proteins using an associated His-tag. We then performed a Bradford assay to determine the protein concentrations and verify that protein production had occurred.

Initial assessments from the Bradford assay showed that buffer lysis did not effectively yield any purified protein while the French Press method showed a very low concentration of purified protein. With the observation that the concentration of protein in the sample was very low, we decided that for subsequent purifications, we would primarily use the French Press lysis method, scale up the volume of our inoculum, and increase the incubation with the IPTG inducer. In an attempt to visualize what little protein we had purified, we ran our purified antigen sample on an SDS-PAGE gel and attempted to visualize using the TGX stain-free method.Unfortunately, were unable to visualize any bands and postulated that it was due to low protein concentrations or improper gel polymerization.

We repeated the protein purification process using a new overnight culture of Rosetta-Gami cells and a larger amount of media for both the Q-sensor and antigen. However, our Bradford assay showed again that our lysate samples did not contain any protein. We ran two SDS-PAGE gels using both TGX-stain free and coomassie blue visualization methods but only faint bands could be seen in the TGX-stain free. We decided to focus purely on the antigen protein and redid the protein purification process once more for that one construct. After the Bradford, we were able to obtain an acceptable amount of purified antigen that was required for the nanobody library screening and would need to run it on a gel to confirm.

Due to the struggles we encountered with the visualization methods of our protein in Week 15, we opted to visualize our proteins using Silver staining, a much more sensitive detection method. We were able to visualize both our antigen and q-sensor protein constructs which were found at the correct bands when compared to the ladder. This was the confirmation we needed to submit our purified Antigen sample for the nanobody library screening process.

Nearing the end of the competition we came to the realization that we would not be able to submit our parts without adherence to the BioBricks standards upheld by the iGEM organization. As our parts did not fall into biobrick standards and we were worried about our protein expression levels under a T7 promoter, we decided to utilize the pJUMP28-1A plasmid from the iGEM distribution kit. Developing a new cloning strategy using gBlocks of our Q-Sensor components and the corresponding regulatory DNA parts would allow us to adhere to BioBrick standards and utilizing a constitutive promoter would allow us to address our inconsistent protein expression results. We prepared for the upcoming gBlocks cloning in pJUMP28-1A by transforming DH5ɑ competent cells with the resuspension from well A10 in the distribution kit, and prepared suitable cultures that were stored as glycerol stocks until we required them for use.

This week we pivoted the focus of the work in the wet lab towards cloning our gBlocks Gene Fragments we had ordered to allow us to adhere to iGEM/BioBrick standards for the submission and characterization of the parts in our Q-Sensor synthetic construct. Our cloning strategy required the use of restriction enzymes and T4 DNA Ligase; thus in Week 17 we began to lay the foundation for the assembly of our parts. We used DreamTaq to amplify the key parts of the Fungalescence construct as well as the regulatory parts that control its expression. We mini prepped pJUMP 28-1A to use as our entry vector as well as the J61002 plasmid containing the J23100 promoter part we wished to utilize as a constitutive promoter. After considerable utilization of the T7 promoter in our pET-22b(+) constructs and navigating certain considerations associated with its usage, we decided to re-engineer our expression system and put our parts under the expression of a constitutive promoter, such as J23100, instead of an inducible promoter such as the T7 promoter. This was because we had encountered issues optimizing the amount of IPTG inducer to add to cultures to result in high protein expression without becoming toxic to cells, and we generally were not observing a significant level of protein production. For the purposes of our project, controlling expression of our constructs with a constitutive promoter was more cost effective (we no longer had to purchase and use IPTG) and was more logical as our goal was to produce proteins continuously for purification, identification, and eventual experimentation. Inducible promoters are advantageous when the goal is to strategically regulate protein expression. We concluded the week by setting up overnight restriction enzyme digestions of the ShadowR, mScarlet, and RBS parts, along with the J61002 plasmid to excise the J23100 part, and the pJUMP vector to excise the empty backbone without sfGFP.

Week 18 focused on continuing the gBlocks cloning experiment to assemble and characterize the parts of our Q-sensor synthetic construct. We began the week by running an agarose gel to verify the digestions from the end of Week 17 were successful and followed up with a gel extraction of the digested parts. Unfortunately this was not our week when it came to gel extractions as we ran into issues visualizing the DNA bands for excision on the UV transilluminator, and obtained mediocre results from said extraction. We now had two unsuccessful gel extractions of the empty pJUMP backbone so we instead experimented with a ‘dirty’ ligation of the pJUMP, RBS, and J23100 promoter parts where we combined the contents of all the reactions together without gel purifying the specific DNA pieces we intended to ligate together. This was in the hope that we would be able to obtain at least one colony with the ligated pJUMP plasmid but ultimately we obtained no white DH5ɑ colonies indicating that no insert other than sfGFP had ligated into our pJUMP backbone. We returned to the figurative ‘drawing board’ and ultimately decided to order a gBlock of a constitutive promoter as we encountered too many issues attempting to purify J23100 from the J61002 plasmid and we were approaching deadlines. While we waited for these parts to arrive, we began brainstorming how to resolve our low ligation efficiency as well as our poor gel extraction results. We closed the week by preparing the new J23101-B0034 (URS) gBlock part for cloning by amplifying it with DreamTaq and digesting it with restriction enzymes. We also re-digested our pJUMP vector with the intention of rectifying the gel extraction issues we encountered with its use in Week 19. Week 18 challenged our ability to identify why a protocol might be unsuccessful, and what steps needed to be taken to fix/modify it for success.

The gBlocks cloning experiment continued into Week 19. The week started by finishing preparations of the pJUMP vector for cloning and setting up our first ligation using the new URS part. Our goal was to ligate pJUMP+URS+mScarlet as well as pJUMP+URS+ShadowR together, transform these ligations into DH5ɑ, perform a colony PCR to verify the inserts, and extract the positive colonies (determined by the results of the colony PCR) plasmids to send for sequencing. Our first attempted ligation results in little/no colonies so we decided to consult with our advisors and redesign our approach. We changed our DNA quantification strategy for ligations by estimating DNA concentration from a gel instead of using the nanodrop spectrophotometer, which can result in inflated DNA concentrations due to contaminants. Unfortunately we continued to battle with low yield and poor quality from our gel extractions. We successfully performed a ligation reaction with the URS, mScarlet, and ShadowR gBlocks, transformed the ligations into cells and observed white DH5ɑ colonies, allowing us to proceed with a colony PCR. The colony PCR reaction allowed us to identify colonies containing our desired insert, which ended the week by extracting plasmids from these positive colonies. We also began preparing the ShadowR and Cameleon gBlocks for the next step in our assembly where our goal was to ligate pJUMP+Cameleon+ShadowR together. The ultimate goal of the gBlocks cloning was to assemble the entire Q-sensor construct (pJUMP+URS+mScarlet+Cameleon+ShadowR) and regulatory regions. The success of the URS+mScarlet ligation in pJUMP brought us to the midpoint of our desired final outcome.

Our final weeks doing experiments in the wet lab had arrived! We began the week by preparing competent SHuffle-T7 cells in the hopes that we would be able to transform them with our successful URS-mScarlet and URS-ShadowR ligations and observe red and grey/purple colonies, respectively, as we had done with our pET-22b(+) clonings. We sent our positive colony pJUMP+URS+mScarlet ligations as well as the pJUMP+URS+ShadowR ligations for sequencing and received our results that confirmed colony 2 and 3 of both the mScarlet and ShadowR assemblies were successful. We attempted to transform both Rosetta-Gami and SHuffle-T7 with the positive colony plasmids but were only successful with SHuffle-T7. From the transformations, the plates were re-streaked to obtain individual colonies which observed distinct red and grey/purple cells, suggesting our redesigned expression system was working properly. The colonies from these plates were further utilized to help characterize the URS-mScarlet and URS-ShadowR for submission. We also prepared and set-up a pJUMP+Cameleon+ShadowR ligation which was ultimately unsuccessful on the first attempt but worked when we made modifications to optimize our T4 Ligase reaction. This ligation reaction was transformed into DH5ɑ and through colony PCR we confirmed the presence of our desired Cameleon-ShadowR insert in transformed colonies. With wiki freeze fast approaching, our team decided we had reached a natural break and concluded our wet lab work. Though we did not complete our initial goal of the gBlocks clonings, we were able to successfully ligate both halves of the Q-sensor construct into the pJUMP plasmid, providing a clear path for the next iteration of lab work. Perhaps one of the most prolonged issues we had faced in our project was also rectified this week, we were finally able to optimize our gel extraction protocol to obtain better DNA yield.

Our final days spent in the wet-lab focused on gathering data for the characterization of our URS-mScarlet and URS-ShadowR composite parts. We designed a straightforward characterization strategy growing the transformed SHuffle-T7 colonies at different temperatures in liquid and on solid media and compared the visible fluorescence of the cells. Check out our parts registry and our results page for an indepth look at the outcomes of this experiment. With that, we concluded our work in the lab for the 2023 iGEM competition!

Between August 23rd and September 1st we performed three rounds of Nanobody library screening which gave us an enriched binder pool. From this we were able to identify potential binders using a fluorescence assay. After fluorescence values were corrected relative to sample ODs we isolated plasmid DNA from a handful of potential binders. We then PCR amplified the Nanobody region from these plasmids and sent them off for sequencing. Leaving us with a final nanobody sequence that we were able to model into a 3D structure.