Project
Human Practices
Dry Lab
Wet Lab
Medal Criteria
In our project, we conducted a series of experiments to assess different parts of our design. We approached our system step by step to gain insights and improve our approach. While our experiments provided insights into different aspects of our design, more technical and biological replicates are necessary to fully characterize the system and ensure the reliability of our data. A larger number of replicates would reduce variability and provide a clearer picture of how our system behaves under various conditions. However, within the time constraints, we tried to gather as much data as possible. Our work provides a starting point for future studies to expand upon.
| Experiment | Short Description |
|---|---|
| Experiment 1: Usage of E. coli BL21 DE3 cells for a plate reader assay | We conducted experiments using E. coli BL21 DE3 cells containing the T7 expression system, which share similar traits with P. putida cells (our Project Design chassis). Our aim was: 1) to identify the most suitable promoter for the T7 polymerase production system, 2) to assess the T7 promoter and the T7phi6 terminator, 3) to evaluate various RBSs for the P3.1 stationary promoter. We measured sfGFP levels and OD600. |
| Experiment 2: Usage of E. coli DH5a cells for a plate reader assay | We conducted experiments with E. coli DH5a cells, which do not contain the T7 polymerase system. Using these cells, we aimed to better characterize our BG37 promoter by testing different sources of carbon and backbones and measuring the differences observed. We also aimed to determine if our T7 polymerase production device functions as a level 2 construct. We measured sfGFP levels and OD600. |
| Experiment 3: Usage of P. putida KT2440 cells for a plate reader assay | We conducted experiments with P. putida KT2440, which is our Project Design chassis. Our goal was to further characterize the BG37 promoter by testing different carbon sources and measuring the observed differences. We measured sfGFP levels and OD600. |
| Experiment 4: Evaluation of gene silencing in Verticillium dahliae | We conducted experiments to determine if we could induce gene silencing in V. dahliae. We produced dsRNA in vitro to treat fungal cultures. We measured OD600 and used RT-qPCR to compare fungal growth and gene expression levels. |
Details about the experiments will be presented here.
| Strains | Genotype |
|---|---|
| E. coli | |
| E. coli DH5a | Δ(argF-lac)169, φ80dlacZ58(M15), ΔphoA8, glnX44(AS), deoR481, rfbC1, gyrA96(NalR), recA1, endA1, thiE1, hsdR17 |
| E. coli BL21 DE3 | F-, ompT, gal, dcm. Ion, hsdSB(rB-mB-), λ(DE3 [lacI lacUV5-T7p07 ind1 sam7 nin5]), [malB+]K-12(λS), pLysS[T7p20 orip15A](CmR) |
| E. coli HB101 | F–mcrB mrr hsdS20(rB–mB–) recA13 leuB6 ara-14 proA2 lacY1 galK2 xyl-5 mtl-1 rpsL20(SmR) gln V44 λ– |
| E. coli DH5aλpir | λpir phage lysogen of DH5α |
| E. coli PIR2 | F-Δlac169 rpoS(Am) robA1 creC510 hsdR514 endA recA1 uidA(ΔMlui)::pir |
| P. putida | |
| P. putida KT2440 | Wild type strain derived from P. putida mt-2 cured of the pWW0 plasmid |
| P. putida Δrnc | Δrnc |
| Vectors | Description |
|---|---|
| pTnS-2 | ApR, ori R6K, TnSABC+D operon |
| pRK600 | CmR, ori ColE1, tra+mob+ of RK2 |
| pTn7-M | KmR, GmR, ori R6K, Tn7L and Tn7R extremes, standard multiple cloning site |
| pUPD2 | CamR, ori pMB1, lacZ gene |
| pDGB3alpha1 | KanR, ori pBR322, lacZ gene |
| pDGB3alpha2 | KanR, ori pBR322, lacZ gene |
| pDGB3omega1 | SpecR, ori pBR322, lacZ gene |
| pSEVA23g19[g1] | KanR, ori pBRR1, lacZ gene |
Throughout our journey, we made sure to keep a detailed lab book to reference our past experiments and tweak any steps we deemed important. When conducting a protocol, each member of the Experimental Wetlab Team took note of their actions. This facilitated the improvement of our experiments and ensured cohesion in our practices, as each member could reference the notes of another member at any time.