Team:Lethbridge HS/Results
At Home composting Study Results
Due to covid, we were unable to do any wetlab work until October. our team took advantage of this, and a group of us began a study of at home composting, in which we each made individual compost bins and studied the results. This page contains our verdict for small scale at-home composting and our progress in the lab.
This season a few of our team members tried out home composting. Through a qualitative approach, we determined the bottlenecks and challenges that a simple composting container can have. For the sake of an indoor type system, we avoided the use of worms that are commonly used in outdoor composting. Through our personal experience with home composting, we determined that the major drawbacks of small scale composting are the effects of a changing climate, the time it takes for decomposition to occur, and how consistently you have to aerate it.
Figure 1: An example of one of the at home composting experiments after three weeks had passed. As seen, very little decomposition has occurred.
Proper composting needs to have the right amount of moisture, oxygen, and carbon: nitrogen material ratios in order to decompose efficiently. If these specific needs are not met, the compost will not decompose at a efficient rate. With our project, organic waste breakdown can be sped up due to pectinases contributing to the decomposition process. As we are building a thermostable pectinase variant, the climate or specifically the heat of the compost will not cause a loss in enzymatic activity.
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Figure 2: The basic factors associated with optimal conditions for promoting efficient organic food waste decomposition
Wetlab Results
Despite loss of time in the lab, our team was able to do some basic molecular biology within the lab. For more experimental details please see our notebook or our experiments page Since we ordered our constructs in pUC57, we were able to immediately transform them into DH5α and BL21. We were able to get 4/6 constructs into DH5α and 6/6 into Bl21.
Figure 3: Bacterial transformations of PelB and PelC constructs into DH5α using chemically competent cells.
Figure 4: Bacterial transformations of Pnl, PelB, and PelC constructs into BL21 using chemically competent cells.
We also attempted to restriction digest the pSB1C3 backbone and our constructs. We successfully digested pSB1C3 as well as 4/6 constructs as seen below.
Figure 5: Restriction digest of pSB1C3-RFP plasmid DNA using EcoRI and PstI enzymes run on a 1% agarose gel.
As shown in the gel, the digestion of the pSB1C3 plasmid samples in Lanes 2, 4, and 5 were successful. The bands corresponding to the RFP gene can be seen at 1000bp and the pSB1C3 plasmid can be seen at around 2000bp. The samples were excised and extracted for future use in DNA ligations.
Figure 6: Restriction digest of PelB and PelC DNA from the pUC57 plasmid using EcoRI and PstI enzymes run on a 1% agarose gel. The red boxes show the band corresponding to that of our DNA construct.
In Figure 6 we can see that plasmids containing PelC, PelB and PelB-GFP cut successfully and that the lower band indicates successful double digests, removing the DNA construct from the plasmid. Upper bands in these samples correspond to single digestions, uncut plasmid and likely supercoiled plasmid DNA. PelC-GFP in lane 4 shows similar results as well as an unexpected band at around 1500bp. we are unsure as to why this band occurred as there are no illegal cut sites when the sequence was double checked.
Future Directions
As seen in our basic experimental flow through, We still have a lot of work to do in the lab. Our next steps are to characterize our thermostable variants and then determine how well our tPectinACE system works on actual compost samples. This will determine how well decomposition is facilitated and if it could be implemented into industrial compost facilities or at home compost systems. For more detailed information please see our modelling and design page.
