Results

With this procedure we were able to have an unlimited supply of our genes and parts of interest without needing to order more.

In order to achieve these transformations we digested the plasmids with restriction enzymes and introduced our parts with Gibson Assembly. To confirm that the transformations were properly achieved we did agarose gels for electrophoresis to identify the band that corresponds to our gene of interest. For this case we also confirmed our positive results with Sanger sequencing and performed the alignments of our sequenced results with our designed parts on Benchling. Since for most of the parts its length was much higher than 1000 bp we used reverse and forward priming to obtain the maximum possible information on our alignment results.

After we obtained the results we did liquid cultures of our samples and saved glycerol stocks and minipreps of them. We also measured the DNA concentration results of the colony PCR purified products and the backbone we wanted to use (p-JET) with the Nanodrop, obtaining the following results:

	DNA Concentration [ng/μL]
Cry 3Aa Sample 1	25.7
Cry 3Aa Sample 2	109.6
Cry 3Aa Sample 3	144.3
Cry 4Ba	224.4
Endolysin Sample 1	60.8
Endolysin Sample 2	155.9
p-JET	69
sfGFP	138.6
mCherry	144

To achieve this, we began with the Gibson assembly to introduce our parts along with our two promoters inside p-END.

After obtaining promising results with the Gibson Assembly and transforming NZY5α E. coli cells, we incubated them at 37°C. However, during this first cycle, we discovered that the transformation containing the MG10 promoter and Cry4Ba toxin constructs did not yield any colonies on any of our plates.

With the grown colonies we performed a PCR Colony and an agarose gel for electrophoresis, it confirmed that some of the constructs had been integrated properly, since the results for the Module 3.1 required more time we sent to sequence the PCR purified products for the parts corresponding to the Module 2 and the Module 3.2 and analyze its results with Benchling.

This consisted in transforming yet again another E. coli strain, that has the particularity of having the same methyltransferase as C. acnes. The goal is to obtain the constructs with a pattern of methylation that ressemblance that of C. acnes.

To start off, we performed minipreps from the liquid cultures of the NZY 5α confirmed colonies, obtaining the following concentrations:

	DNA Concentration [ng/μL]
Cry3Aa+MG10	80.4
Cry3Aa+MG26	137.4
Cry4Ba+MG26	255.6
Endolysin+MG10	54.8
Endolysin+MG26	151.3
sfGFP+MG10	106.5
sfGFP+MG26	109.5
mCherry+MG10	149.9
mCherry+MG26	101.4

After transforming the E. coli EC24 strain with the minipreps and culture overnight, once again we performed PCR colonies, in order to send the purified products to sequence. The colonies were again confirmed through alignment, the analysis of these sequencing can be seen on the document attached below.

For this experiment, the EC24 strain of E. coli was transformed with plasmids encoding two fluorescent proteins. The transformed E. coli cells were grown in liquid culture at different temperatures (room temperature, 30ºC, and 37ºC) to assess the effect of temperature on protein production. Fluorescence intensity was measured two days after for 30ºC and 37ºC, and we waited two more days to measure the room temperature sample, to assure correct growth. By doing so, we quantified the expression levels of the fluorescent proteins. We anticipated that higher temperatures would lead to increased fluorescence, reflecting the activation of transcription via the RNA thermometer.

Just by looking at the liquid culture growth, it was apparent that temperatures close to 37ºC had significantly more protein production, in comparison to those that had grown at room temperature and 30ºC.

Nevertheless, we also performed a quantitative analysis of the amount of fluorescence in all the samples. The protocol was straightforward: after incubating the transformed E. coli EC24 cells at different temperatures, we transferred aliquots of each culture into a 24-well plate and measured fluorescence using a plate reader. For GFP, the excitation and emission were measured at 485 nm, while for mCherry, it was measured at 575 nm. The absorbance was also measured in all samples at 660 nm.

To ensure accuracy, all fluorescence readings were normalized by applying a simple formula:

Sample reading (sfGFP/mCherry) - LB reading (sfGFP/mCherry) Sample ABS 660 - LB ABS 660

The LB served as the blank and this normalization allowed us to account for the background fluorescence and ensured that the fluorescence values correspond only to the expression of the different proteins.

In both cases, there is a significant difference between higher and lower temperatures. When comparing 30ºC and 37ºC, there is around a two-fold difference in each case, proving the functioning and ideal working temperature of the FourU RNA thermometer (at least in E. coli).

Note: we measured the fluorescence of each liquid culture three times in order to have replicates.

As mentioned, the first couple of transformations were a failure due to both inexperience and the intrinsic difficulty of correctly transforming C. acnes. After these two first attempts, we finally succeeded in introducing our first construct into C. acnes: the CAP 10-3 Endolysin.

As can be seen in the gel, apparently all of the constructs had been correctly integrated. However, the latter 16S region analysis through alignment (see C. acnes alignments pdf), would show that C. acnes was only present in the endolysin samples. The other “positive” samples were Staphylococcus species that had been able to incorporate our construct.

Successfully obtaining the correct transformations in C. acnes was a crucial step for our project. After many setbacks, this achievement opens up the possibility for further experiments that couldn't be finished before the iGEM wiki freeze. Moving forward we can focus on testing the remaining constructs and exploring additional applications. This progress gives a strong foundation for future work and ultimately, the development of our final product.

The samples analyzed included:

For E. coli, proteins are primarily intracellular, requiring cell lysis to isolate the proteins from the pellet. For C. acnes, we anticipated protein expression both within the cell and in the supernatant. Thus, we evaluated both pellet and supernatant samples to capture the full protein profile.

To ensure consistency, we loaded 20 ng of total protein per well, adjusting sample volumes based on protein concentration. Before loading in the samples, we evaluated the concentration of protein found in each one to know how much was needed to have the 20 ng.

Sample	DNA Concentration [ng/μL]
E. coli Cry3A+MG10 (pellet)	1.179
E. coli Endolysin+MG10 (pellet)	1.499
C. acnes Endolysin+MG10 (pellet)	0.512
C. acnes Endolysin+MG26 (pellet)	0.692
C. acnes Cry4Ba+MG26 (pellet)	0.745
C. acnes Endolysin+MG10 (SN)	0.686
C. acnes Endolysin+MG26 (SN)	1.072
C. acnes Cry4Ba+MG26 (SN)	1.018
Control sample	1.32

Once we have obtained the total protein concentrations, we can proceed with the results of the gel:

We have also attached an image of the ladder used for reference. For reference, the expected kDa were the following:

Results

After revealing the Western blot, we noted the following:

In conclusion, protein expression was confirmed for E. coli Endolysin+MG10 and C. acnes Endolysin+MG26. Protein expression is concluded to also apply for E. coli Cry3Aa+MG10. We would have liked to perform another Western Blot with newly transformed C. acnes cells, however, we did not have enough time to do this.

To determine the effectiveness of the CAP 10-3 endolysin in reducing C. acnes viability, we compared the growth of three different strains: one expressing an endolysin-coding plasmid, one with a plasmid providing only antibiotic resistance (WT), and another one with a plasmid with constitutive GFP expression. Growth was monitored over the four following days by measuring the OD600. The OD600 of the blanks was subtracted from all the experimental readings to account for background absorbance.

This indicates that the expression of endolysin, regardless of the promoter used, is effective in impairing C. acnes viability. The consistent reduction in growth across strains with different promoters suggests that endolysin has a significant impact on its overall fitness.

The endolysin-expressing strain exhibited a lag phase that was noticeably longer than that of both the WT and GFP strains. This extended lag phase suggests that the presence of endolysin may require additional time for the cells to acclimate before starting to grow. Once the lag phase concluded, the growth rate of the endolysin strain appeared slightly slower than that of the WT and GFP strains. The OD600 measurements indicated that, although the endolysin strain eventually increased in cell density, it did so at a reduced rate, further supporting the fact that endolysin affects cellular viability.

These findings are important as they support the idea that endolysin may assist in the delivery of the Cry proteins. Future research should focus on investigating the specific role of endolysin in facilitating protein release, and evaluate whether this mechanism enhances the therapeutic effectiveness against the targeted skin infestations

The endolysin has been essential to our project, playing a key role in both ensuring the safe use of our GMO and facilitating the release of the Cry protein. We are particularly proud of the endolysin DNA part, which we are presenting for the Best New Part special prize due to its innovation and importance in our design. You can find more details about the part in the iGEM registry here.