Throughout our project we were able to obtain both positive results, recognize the mistakes we made along the way, and identify aspects for improvement for the future. Our commitment to scientific research has illuminated not only the successes we celebrate today but also the paths toward refinement and the exciting possibilities that lie beyond.
Transformation Results
One of the positive aspects of our results is that we were able to obtain transformed cells for our Ampicilin resistance plasmid with BBa_K4881027 insert (Construct 1), and two kanamycin resistance plasmids with BBa_K4881028 (Construct 2), and BBa_K4881029 inserts (Construct 3). In addition to that, we obtained some transformed cells with the plasmid BBa_J04450 (O11) from the iGEM 2023 DNA Distribution Kit, which would serve as the cloramphenicol backbone of our third construct. Pictures of both our E. coli cells in petri dishes and liquid media can be seen at the side.
Following the transformation process, a noticeable issue emerged as the transformed cells failed to express chromoproteins — a pivotal indicator for the incorporation of the plasmid into the cells. The absence of chromoprotein expression strongly implies problems in the successful integration of the reporter genes into the inserts of the plasmid. Exploring possible optimization measures for our insert will be essential to ensure the complete success of future transformations, achieving the intended functionality of the reporter gene, and the improvement of our biological parts.
DNA Extraction and Gel Electrophoresis Results
After obtaining the liquid culture of the transformed cells, we attempted to perform DNA extraction. In this way we could corroborate if the plasmid had been correctly integrated into the E. coli cells through a subsequent setup of the DNA in electrophoresis gel.
A positive outcome of the electrophoresis gel is that the bands corresponding to the two samples of the plasmid with Construct 2 manifest between the HindIII Digest lines spanning 4,361 to 6,557 base pairs. This alignment is consistent with expectations, considering that our insert, BBa_K4881028, of 3,801 bases resides in a kanamycin resistance plasmid measuring 2,705 base pairs. Therefore, we can infer that Construct 2 was most likely taken in by the cells. However, a comprehensive conclusion is hindered by the absence of electrophoresis lines for the IDT Construct 2 DNA.
The absence of detectable DNA bands in most of our cells, the IDT constructs, and the iGEM delivery kit raises questions about possible deficiencies in our electrophoresis gel setup or the DNA extraction process. The use of disinfected parafilm for DNA staining during electrophoresis is the most notable concern, as it may have resulted in DNA drying out due to environmental exposure. While replicating this experiment in the future, we will follow the proper protocol and avoid making mistakes that could potentially damage our results.
This season we found ourselves unable to replicate DNA extraction and electrophoresis due to the time constraints. Therefore, to check the expression of the PETase and MHETase proteins with construct 1, we proceeded to do a bioassay to evaluate the plastic degradation caused by our bacteria.
Bioassay Results
For our 72-hour bioassay setup, we used 15 autoclaved Falcon tubes of 50mL, 250mL autoclaved liquid LB, 250mL autoclaved liquid LB + Amp, 250mL autoclaved liquid LB + Kan, 250mL autoclaved liquid LB + Amp+Kan, and 3 mL of liquid culture of each type of bacteria to use. The plastic samples came from a PET bottle pierced with an office hole puncher. Afterward, the PET circles were weighted in groups of 6 to be 0.056 ± 0.002 g. Then, they were treated in 70% ethanol for 15 minutes and washed with distilled water. The liquid culture for the bioassay contained 500µL of overnight culture to 5mL of media placed on 50mL Falcon tubes for aeration. After that, we placed 6 PET circles inside the tubes. Then, 20 hours after the bioassay tubes were closed, we added 5mL of media. Finally, they were left until the end of the 72-hour period.
The results of our bioassays reveal a promising aspect: the plastic degradation of 5 to 7% in the tubes containing the E. coli cells with Construct 1. Therefore, we can be positive that our construct has a certain level of efficacy. However, it is crucial to recognize the challenges inherent in our experimental setup, particularly a significant margin of error due to the scale of the experiment. The low mass of the plastic samples and the limited volume of liquid culture raises concerns about the accuracy of the results, especially when analytical balances are used. This can be evidenced by the subtle mass changes of 0.001 g in other samples. To strengthen the reliability of our findings, future experiments may benefit from a larger sample size.
An intriguing aspect of our results leads us to explore the relationship between construct 1 and construct 2, specifically questioning how construct 2 may be influencing bacteria with constructs 1-2. Given the critical role of Construct 2 in ethanol production, we hypothesized that the ethanol generated could be affecting cell survival, potentially hindering plastic degradation.
Finally, we believe that the ability of bacteria to degrade plastic samples was not fully explored because the plastic circles in the samples adhere to each other, significantly reducing the overall surface area available for bacterial interaction. Something that made this happen is the shape of the Falcon tubes, so when all the circles sank, they ended up stacking on top of each other. Therefore, when replicating this experiment in the future we want to use airtight, autoclaved 100 ml bottles. This refinement of the procedure eliminates the previous problem and provides the additional benefit of accommodating larger volumes of liquid cultures and plastic samples. Thus raising the scalability and precision of our experimental replication in subsequent research.