Results

Overview of Accomplishments

Produced and characterized endo-protease necessary for keratin degradation within E. coli
Produced and secreted GFP using our pBbB8k plasmid construct and the curli secretion pathway
Suspended E. coli within an alginate gel to prove the viability over a period of time. Further, we induced the bacteria while they were suspended and demonstrated successful secretion.
Showed proof of concept by inducing bacterial colonies within an alginate suspension to secrete GFP
Tailored product design to consider a biologically safe alternative, replacing IPTG with Arabinose. Experiments were successful in both of these systems.
Developed a protocol for easy alginate gel formation and the encapsulation of cells within the gel. We hope other iGEM teams will find this protocol under our Protocol Tab!

Endo-protease 6877

The following results were obtained after inducing our transformed DH10b + Gibson construct cells.

Results of measuring A280 by use of nano-drop

The results obtained from the nano-drop indicated that culture 2 had been successfully assembled and induced, while culture 1 had not. To confirm these results, we ran SDS page with both induced constructs alongside our controls.

GFP production and secretion

As a proof of concept to show that our construct could both produce and secrete our proteases, we used our pBbb8k vector and construct to produce and secrete GFP in our cells.

The graph above was obtained using data from a SpectraMax M2 plate reader. For this test, we used a culture of E.coli Nissle obtained from the Wyss Institute that utilized the same curli secretion pathway to secrete GFP as we were using to secrete endo-protease. We can see that our induction worked extremely well, since the relative florescence of our 2 induced forms (centrifuged and not centrifuged) were significantly greater than our blank and non-induced control. Furthermore, we see a higher relative florescence in the wells with cells and supernatant than the wells with only supernatant (this is expected since some GFP is likely trapped within the cells). When we spin the cells down and measure only the supernatant, we still see a relatively high florescence compared to the non-induced control and the blank, meaning that although there was some GFP still in the cell as well as GFP secreted outside of the cell by the curli secretion pathway.

The graph above was obtained using data from a SpectraMax M2 plate reader. For this test, we used a culture of chemically competent BL21 that we transformed with our GFP curli secretion construct. The results are extremely similar to the GFP Nissle secretion test, showing that we successfully transformed our vector into our new cells.

Alginate formation and cell encapsulation

The next step was to show that we could successfully encapsulate our engineered cells inside of an alginate gel, keep them viable, and induce them from outside the gel. The first step in this process was to delineate a procedure for forming an alginate gel and encapsulating cells within.

gels can be seen in the top 3 rows of the left-most and right-most columns

The above wells were created by following the alginate cell encapsulation protocol found in the protocol section of our wiki. Although not conducive to uniform gels, this protocol lends itself extremely well to quick and easy cell encapsulation. We then used these gels for further testing, such as the florescence testing we carried out in the section above.

After forming our alginate gels containing our engineered cells, we hydrated them with media. Before storing them, we attempted to induce half of the gel beads by adding Arabinose to the media surrounding the bead. The images below were obtained the morning after induction and a night of incubation.

TOP10 cells w/ pBbb8k-vector in Alginate - induced with arabinose 100x oil objective (Zeiss EC Plan-NEOFLUAR), 150 ms exposure Zeiss Axio fluorescent microscope

TOP10 cells w/ pBbb8k-vector in Alginate - uninduced 100x oil objective (Zeiss EC Plan-NEOFLUAR), 150 ms exposure Zeiss Axio fluorescent microscope

We can clearly see that we were successfully able to induce our cells from outside the patch. To get more quantitative data, we again performed a secretion test using the SpectraMax M2 plate reader.

The non-induced cultures are plotted on the left, and the induced cultures on the right. All fluorescence has been normalized to the mean fluorescence of the non-induced colonies, so our graph shows the relative fluorescence intensity of each culture, relative to the mean fluorescence of the non-induced cultures. From this graph we can clearly see that we had successful induction of our cells within the alginate, with roughly a 4-fold increase in relative mean florescence intensity between our induced and non-induced culture.

Results Analysis and Summary

What do our results actually mean in terms of our ideal product?

Throughout our work this summer, our goal was to attain data sufficient to show that a keratinase patch, such as the one we suggested, would be a technically feasible product. To do this, we strove to show the production and secretion of the proteases necessary for complete keratin degradation was possible with E. Coli, and that these protease secreting cells could be successfully encapsulated and induced within alginate. The results we have attained begin to point toward such a conclusion. Our initial PCR results told us that we had indeed successfully replicated our plasmid backbone while taking out the intended portion, allowing us to continue with Gibson assembly. After Gibson assembly, a second gel image confirmed that we had successfully inserted our construct into our plasmid. Our results from the protein gel, obtained after inducing a culture of E. Coli with our altered plasmids, (hopefully showed us that we successfully produced our protein). The GFP secretion tests that we ran in parallel confirmed that utilizing the curli secretion pathway was a viable option for the secretion of proteins produced within the cell, and that our cells could be induced even while inside an alginate solution. But what does all of this mean in terms of a potential product?

These results show that there are grounds for an alginate patch containing E.coli, inducible by a secondary application of arabinose, that could produce and secrete keratinase. Although this product would require further research and testing, some of which is highlighted in our “unsuccessful results” section below, we feel as though our successful results lay the groundwork for a positive proof-of-concept.

Unsuccessful Experiments

Where do we have opportunities to learn and grow?

Cloning

As is common with PCR, our protocols required fine tuning. Our initial runs had extremely low yields, and it took time to optimize our protocol to produce the yields we wanted. This obstacle is frequently associated with complex cloning, but this experience will helped us to better understand the cloning process, and will allow us to more easily troubleshoot this process in the future.

Part synthesis

Some of the parts we ordered were delayed and ultimately ineffective due to CG repeats in their sequence. This delay not only increased our time strain, but also forced us to change our experimental designs. Although this was a good practice in adaptation, this is an obstacle we have learned to spot, and more easily avoid in the future.

Team:Harvard/Results