This week marked our first official week in the lab for the NU iGEM Team. We planned to begin growing bacterial cultures of Luke-2, RFP, pET28a_CBM_CohIII, and pET28a_Xyl_Doc. However, since only the RFP plasmid had arrived, we proceeded with that while waiting for the remaining constructs to be delivered. To support upcoming experiments, we prepared fresh kanamycin plates and aliquoted several antibiotics, including kanamycin, ampicillin, and chloramphenicol for future use. We also made bacterial growth medium and LB agar to establish baseline materials for the team. Finally, we performed plasmid purification on the RFP plasmid. The nanodrop measurement showed a concentration of 123.65 ng/µL, which gave us a great starting point for subsequent experiments.
This week, we purified the RFP plasmid. Initially, plasmid DNA was amplified via polymerase chain reaction (PCR) using the Q5 2× Master Mix to generate multiple copies of the RFP sequence. The PCR products were subsequently purified using the Monarch PCR purification kit, followed by agarose gel electrophoresis to confirm the presence of the RFP plasmid. The purified products were then quantified using a spectrophotometer. In addition, the pET28a plasmid was transformed into E. coli BL21DE3 cells.
We repeated the transformation of the pET28a plasmid into E. coli DH5a cells. Following transformation, the cultures were plated using either 100 µL, 200 µL, or 200 µL of resuspended culture. After overnight incubation, individual colonies from each plate were selected and inoculated into separate cultures for further growth.
We initiated cultures of pET28a-RFP in BL21(DE3) cells by picking clones from a transformation plate. In preparation for creating an RFP iGEM logo, we poured media into the plate design and incorporated LB agar containing IPTG. The pET28a-RFP plasmid was also purified. The logo plates were then streaked with the RFP culture, and fluorescence imaging was performed to test RFP expression and logo visualization.
We transformed Puc19 as a control and plated the cells on ampicillin plates. In addition, we prepared another agar plate with the iGEM logo, streaked it with RFP, and imaged the resulting plates. We also conducted preliminary burn tests using cotton coated with different concentrations of DNA. Finally, overnight cultures were prepared for use in the following week.
We transformed pET28a_CBM_CoHIII and pET28a_DocIII_RFP into BL21(DE3) cells. Following transformation, 100 µL, 200 µL, or 200 µL of resuspended cultures were plated onto kanamycin plates and incubated overnight. Colonies from each plate were then selected to initiate 50 mL cultures. Also, we performed a back-dilution of Puc19.
To assess protein expression, we generated a protein production growth curve by measuring OD values of the CBM and RFP cultures every 30 minutes over a six-hour period. Protein binding activity was also evaluated by submerging cotton samples into supernatants containing either CBM-CoHIII, RFP-DocIII, a mixture of both, or Triton X-100 alone. We also ran some flame-resistance tests of cotton coated with either protein or DNA, measuring how long it took for the cotton to fully burn.
This week, we started cultures of several different carbohydrate-binding modules. We transfected 50 mL cultures with either Luke-2 (with ampicillin selection) or pET28a-CohII-CBM-HIS-YBBR (with kanamycin selection) and incubated them at 37 °C. We also picked colonies from the pET_CBM_CoHIII and pET_RFP_DocIII plates to start three 50 mL cultures of each construct (six total). After sufficient growth, we prepared glycerol stocks of each construct and stored them at -80°C. Additionally, we performed a PCR of pET28a, confirmed the product via gel electrophoresis, and completed a PCR cleanup to generate enough plasmid for next week's Gibson assembly.
We constructed five different plasmids using Gibson assembly with pET28a as the backbone and Alpha-S-Casein, Beta-Lactoglobulin, PurA, Soybean Glutenin, or Streptavidin as inserts. The assembled plasmids were then transformed into competent BL21 cells and plated for incubation. In parallel, we initiated additional 50 mL cultures of Luke-2 and CBM-CohIII-YBBR-HIS, which we incubated overnight and then did a plasmid prep. Following incubation, the transformation plates showed limited growth. So, we picked clones and performed colony PCR only on the three constructs that displayed some growth (Alpha-S-Casein, Soybean Glutenin, and Streptavidin). We also repeated the transformations for all the constructs, this time using 10 µL of culture instead of the 2 µL used previously.
We prepared more Puc19 cultures. We also re-ran a protein gel of CBM-CoHIII and RFP-DocIII, and started more cultures of pET_RFP_DocIII and pET_CBM_CohIII. Then, we ran a PCR on Pet28a. Additionally, we measured a protein growth curve of the RFP-DocIII cultures and CBM-CoHIII cultures, both induced and non-induced (four cultures total).
This week the team focused on maintaining and preparing bacterial cultures. Early in the week, 5 mL of the existing culture was transferred into a labeled 500 mL flask containing 45 mL of LB media and 50 µL of kanamycin to sustain selective growth. Cell density was monitored by measuring OD600 on the NanoDrop. Throughout the process, detailed notes were recorded on setup and handling procedures to ensure reproducibility and streamline future culturing work.
Protein samples were analyzed by SDS-PAGE using pre-cast polyacrylamide gels run at 180V for 1.5 hours, followed by Coomassie Blue staining and gel imaging. Colony PCR was performed using OneTaq MasterMix with construct-specific thermal cycling conditions (94°C denaturation, 49°C annealing, 68°C extension) to screen transformants containing AlphaSCasein, Soybean Glutenin, BetaLactoglobulin, Streptavidin, and PurA constructs. PCR products were analyzed on agarose gels at 140V for 30-45 minutes. Plasmid DNA was isolated from overnight cultures and quantified by nanodrop spectrophotometry. Bacterial transformations were conducted using standard heat shock protocol (30 min on ice, 42°C for 10 sec, 5 min on ice) followed by Super Optimal broth with Catabolite repression recovery at 37°C for 1 hour and plating on selective media.
Alpha-S-Casein constructs were submitted for sequencing and Gibson assemblies were performed using Q5 2x Master Mix with optimized ratios, followed by overnight DpnI digestion at 37°C. Transformations utilized NEB 5-alpha competent cells with heat shock protocol, and protein expression analysis was conducted using BL21(DE3) strains with IPTG induction at mid-log phase. Growth curves were monitored by OD600 measurements and glycerol stocks were prepared for long-term storage. Colony PCR verification employed OneTaq MasterMix with products analyzed on 1% agarose gels.
Protein expression analysis was conducted using SDS-PAGE with cell pellets resuspended in PBS and prepared with Laemmli buffer at 95°C. Gels were run at 100V for 10 minutes followed by 150V for 50 minutes, then stained with Coomassie Blue. Plasmid preparations were quantified by nanodrop spectrophotometry and submitted for sequencing with appropriate sample protection. Transformations utilized BL21(DE3) competent cells with heat shock protocol (30 min ice, 42°C for 45 sec, 2 min ice) followed by SOC recovery and plating on Kanamycin-selective media. Growth curves employed OD600 monitoring with IPTG induction at appropriate cell density, tracking both induced and non-induced conditions for comparative analysis. Alternative incubation temperatures (4°C, -20°C) were successfully implemented when standard ice incubation was unavailable.
This week we analyzed protein via SDS-PAGE of 3-hour samples from five constructs (ASC, BLG, SBG, PurA, Strep) under induced and non-induced conditions, and transformation of Trans7 and Trans5 DNA constructs into BL21DE3 cells. Key issues included improper SOC storage at room temperature instead of 4°C and insufficient gel wells requiring additional runs for Strep and PurA spindown samples. Transformation plates were prepared with kanamycin selection and gel imaging was completed successfully.
This week encompassed multiple molecular biology techniques including transformation verification, protein analysis, PCR amplification, and Gibson assembly preparation. Transformation plates from Week 14 showed successful colony growth and were moved to 4°C storage, followed by bacterial stock preparation using glycerol. ASC protein samples from August 9th were analyzed via SDS-PAGE, focusing on 3-hour time points under induced and non-induced conditions. PCR amplification of pET28a plasmid was performed using iGEM-004/006 primer pairs, followed by DpnI digest for template removal. The week concluded with preparation for Gibson assembly, including gBlock reconstitution and plasmid quantification.
This week we focused on protein characterization and cloning procedures through SDS-PAGE analysis and PCR amplification. Seven ASC protein samples from August 9th (Pre, 1 Hr I/NI, 2 HR I/NI, and 3 HR I/NI) were analyzed via gel electrophoresis after preparation with Laemmli buffer containing β-mercaptoethanol, with samples boiled at 95°C before running at 150V for one hour. PCR amplification of pET28a plasmid was performed using iGEM-005/006 primer pairs with Q5 2x Master Mix, running 28 cycles at 62°C annealing temperature. Following PCR completion, DpnI was added for overnight digestion at 37°C to remove template DNA. The week concluded with gel staining overnight and storage of both protein samples at -20°C and PCR products in the thermocycler for subsequent processing.
This week we focused on troubleshooting protein expression issues through colony PCR verification and protein gel analysis. Due to inconsistent protein gel results, the team initiated diagnostic procedures by streaking five bacterial strains (iGEMS_024 through iGEMS_028) onto Kanamycin-50 plates. Colony PCR was performed using iGEM25-121 and iGEM25-151 primers with OneTaq MasterMix, running strain-specific cycling conditions for ASC, β-lactoglobulin, streptavidin, soybean glutenin, and PurA. DNA gel analysis confirmed plasmid presence in all samples. Additionally, two gBlocks (Cipa_RFP and CbpA_RFP) were reconstituted to approximately 11-12 ng/μL for Gibson assembly preparation with pET28a backbone. The week concluded with SDS-PAGE analysis of protein samples from various time points (Pre, 1HR I/NI, 2HR I/NI, 3HR I/NI), comparing both regular and centrifuged samples to assess protein expression levels. An iGEM_RFP culture was also initiated for future experiments.
This week we focused on Gibson assembly optimization and temperature-dependent protein expression studies. Gibson assembly was performed using pET28a backbone (13.7 ng/μL) and CbpA_RFP gBlock (11.93 ng/μL) with NEBuilder Master Mix, incubated at 50°C for one hour. Following cleanup with NEB PCR purification kit, the assembly product was transformed into DH5α competent cells and plated on Kanamycin-50 plates. Protein expression optimization involved testing iGEMS_024 cultures under varying conditions: two temperatures (32°C and 37°C) and three induction states (1mM IPTG, 250μM IPTG, and non-induced). Growth monitoring over six hours revealed differential expression patterns, with 37°C cultures showing higher OD600 values (reaching 2.74 for non-induced) compared to 32°C conditions (maximum 2.60). Hourly samples were collected, centrifuged, and stored at -20°C for subsequent protein analysis. The week's work established temperature and IPTG concentration parameters for optimizing protein expression in the system.
This week we continued optimizing our Gibson assembly and comprehensive protein expression analysis under varying conditions. Multiple attempts at Gibson assembly were performed using pET28a backbone and RFP_CbpA gBlock, followed by transformation into DH5α cells. Protein expression studies compared iGEMS_024 and iGEMS_025 strains at two temperatures (32°C and 37°C) with different IPTG concentrations (250μM and 1mM). SDS-PAGE analysis of 2-hour and 3-hour timepoints revealed variable expression patterns across conditions. BCA assay was performed to quantify protein concentrations using ethylene glycol-diluted albumin standards, with samples normalized to OD 10. The week concluded with PCR product cleanup yielding concentrations of 81.9 ng/μL and 145.7 ng/μL with good purity ratios. Additionally, PUC19 glycerol stocks were initiated as backup cultures, and transformation plates showed colony growth requiring further screening.
This week we ran a DNA gel, SDS-PAGE gel, transformation into DH5α cells, and harvesting cells for protein extraction. We began by running and imaging a gel of both samples of pET28a. A NEBuilder HiFi reaction was performed using pET28a and Cipa, and an SDS-PAGE gel was run using samples from August 9th. These samples included StrepPre, Strep1HR NI, Strep1HR I, Strep2HR NI, Strep2HR I, Strep3HR NI, Strep3HR I, Strep Pre Sup, Strep1HR NI Sup, Strep1HR I Sup, Strep2HR NI Sup, Strep2HR I Sup, Strep3HR NI Sup, and Strep3HR I Sup. The gel was left to stain overnight, and the results of the HiFi reaction were transformed into DH5α cells. Serial dilutions were performed during the protocol and plated for overnight incubation. Overnight cultures of iGEM_024 and iGEM_025 were also created for later use. These cultures were then back-diluted into fresh LB with kanamycin, and once OD600 measurements reflected growth of 0.22 and 0.19, respectively, they were induced with IPTG. Both non-induced and induced cultures were harvested by centrifugation, resuspended in 0.5% Tween20, and lysed using a NaOH and cellulose solution. They were then treated with ethylene glycol, spun down, and the supernatant was collected and stored.