iGEM 2023 — Team wvhs-sandiego

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## Summary

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All lab work was completed at the J. Craig Venter Institute under the supervision Dr. Erin Garza and Dr. Jocelyn Macho. This page contains a timeline of our in lab work along with what actions were completed. Our process of loop assembly included a mix of Gibson Assembly and Golden Gate Assembly to create L0, L1, and L2 plasmids.

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## Summary

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All lab work completed was done at the J. Craig Venter Institute under the supervision of Dr. Erin Garza and Dr. Jocelyn Macho. The following page includes all experiments and procedures conducted in lab, as well as a brief overview of their purposes in our project. Our assembly methods were loop assembly that utilized both Golden Gate and Gibson Assembly to combine our pL0, pL1, and pL2 parts.

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## Results

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This project was created with the aim to successfully introduce two enzymes, PETase and MHETase, into the marine bacteria: Alteromonas macleodii. In doing so, the enzymes will work sequentially to break down polyethylene terephthalate (PET) plastics. This would be done by first having PETase break off plastic polymer chains from larger sheets and then having MHETase break down those chains into terephthalic acid and ethylene glycol.

Our results showcase that the enzymes, PETase and MHETase, were successfully implemented into Alteromonas macleodii. Reaction 2 which contained joint L1’s with both MHETase and PETase sequences was successfully transformed into E. Coli. From there we conjugated the Level 2 plasmids from E. Coli into the Alteromonas macleodii.

## Successes and Failures

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Our L0, L1, and L2 plasmids were all successfully created inside of the E. Coli. We also managed to conjugate our L2 plasmids with the combined PETase and MHETase sequence into the Alteromonas bacteria.
We had to spend a lot of time redoing our level one plasmids because of issues with our original terminator sequence. Over the years, it had degraded in the Golden Gate assembly but we replaced it with the T1 E. coli rrnB terminator and our colonies ended up growing fine. We originally thought the issue was with our J23 T7 promoter, but we continued using it as that one did work. We also used an inducible Rhamnose promoter for our MHETase plasmid which worked out.

## Errors

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Many of the colonies we attempted to cultivate were very slow growing and when grown looked stressed and elongated. While this could be due to a multitude of issues, we believe that there may be an issue with the secretion enzyme that is supposed to aid with the emittance of the PETase and MHETase enzymes. This could cause an abundance of the enzymes to get stuck in the cells and would contribute to the elongated structure. It would also explain the spots of fluorescence seen in the L1 cultures where it appeared that the plasmids were being grouped together within the bacteria. On top of that, the original terminator we attempted to use (B0015) had degraded over time causing our L1’s not to grow at first. The process of figuring out which part was causing issues took several weeks and set our project back so that we were not able to test the altered bacteria against PET plastics.

## Further Research

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In the event that we continued our project we would re-optimize the MHETase sequence for E. coli and change out the secretion peptide. This would prevent the cells from getting stressed out and accumulating DNA. The next step in our project would be to test the altered bacteria under various temperatures of water to see the impact of the heat stability done by our Dry Lab team.
We would also begin further testing with sheets of PET plastic to see how much was being degraded and analyze the byproducts. As our lab process to introduce the level 2 plasmid into the Alteromonas took longer than expected, we were not able to spend as much time with the plastic degradation aspect of our project. We would also optimize the enzymes secreted in order to degrade as much plastic as possible and measure the amount that has degraded by using weight.

## FTIR Results

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Using an FTIR (Fourier Transform Infrared) machine to analyze the excitation of molecules in the plastic, we were able to get a rough picture of the chemical compound of the plastic with and without exposure to our altered bacteria. Depending on the light received back after flooding the plastic with infrared light, we could see how much of each aspect of the plastic’s makeup was still present. In the control sample with wild type bacteria, the far right of the graph is fairly flat; however, in the week one samples of experiment 1 through 3, there is a peak that can be seen on the far right of the graph. This peak signifies an increase in alcohol groups in the PET plastics. The increase in alcohol groups comes from the breakdown of the plastic and the formation of by-products thus proving the success of our altered Alteromonas when degrading PET plastics.

Here are the FTIR results for our control sample. Notice the relatively flatness at the far-right.

![](https://static.igem.wiki/teams/4730/wiki/ss-1.png)

Here are the FTIR results of the PET plastics with our altered Alteromonas colony. Peaks are evident on the far right for all three experiments.

![](https://static.igem.wiki/teams/4730/wiki/ss2.png)
![](https://static.igem.wiki/teams/4730/wiki/ss-3.png)
![](https://static.igem.wiki/teams/4730/wiki/ss-4.png)
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