We confirmed that lysis occurs by induction of the lytic genes.

Fig.2 Conceptual diagram of our gene circuit

We first tried to see if the lytic gene actually works. For this purpose, we designed plasmids encoding the lambda phage lysis cassette (BBa_K4655006) and the lysis gene ColE7 (ColisinE7) (BBa_K4655007), both regulated by araBAD promoter, and introduced them into E. coli [5]. Subsequently, for each of the lytic genes, we confirmed by measurement of OD600 that the growth of E. coli was restricted when araBAD promoter was activated by the addition of 0.2% arabinose, confirming the occurrence of lysis.

Fig.3 Growth curves of four cultures of E. coli with each of the two lysis genes (lysis cassette of lambda phage and colE7) when cultured at 37°C with shaking in a medium with (+) or without (-) 0.2% arabinose.

When induced with arabinose, the increase in OD stopped even when both lysis genes were used. However, it began to increase again after 6 hours.

In this experiment, the growth of E. coli was observed to stop at around 3~5 hours after the addition of arabinose, which is consistent with Siegele & Hu's (1997) [6] findings about the araBAD promoters. Surprisingly, we found that the population began to rise again after 6 hours. Since this phenomenon was observed in more than one experiment, we speculated that mutants that do not lyse appeared at a high frequency and became dominant in the culture. From these experiences, we realized that a way to prevent the emergence of mutants was necessary.

Development of a strictly controlled lux promoter

Fig.4 Conceptual diagram of the gene circuit where AHL and LuxR combine to activate the mutated lux promoter

When attempting to create a plasmid carrying the lux promoter-controlled system for regulating lytic genes and culturing the transformed E. coli. culture, we obtained a culture with increased viscosity due to lysis, even though it was not induced by AHL. We believed that this was due to leakage of the lux promoter.

Unintended lysis due to lux promoter leakage imposes strong and constant selective pressure within the culture, promoting the emergence of mutants lacking the lytic function. We interpreted the results of our experiment as the proliferation of non-lysing mutants during cultivation.

Considering these results, we concluded that it is necessary to search for or develop a more tightly controlled lux promoter. Through this investigation and experimentation, we confirmed that using mutants 5 and 9 of Part:BBa_R0062, as reported by Team: Tsinghua, resulted in the reduction of the lux promoter leakage, as measured by the fluorescence of sfGFP.

Fig.5 The mutations in the Lux promoter

Mut5 has a G to T change at the -36 site, and Mut9 has a T to G change at the -37 site.

Fig.6

(a) The graph of Fluorescence*/OD600 representing the lux promoter leakage for the original, Mut5, and Mut9 when not induced by AHL.
(b)The graph of Fluorescence* / OD600 when sfGFP expression was induced by the lux promoter for the original, Mut5, and Mut9 at an excessive AHL concentration of 100 nM. Fluorescence* / OD600 at 0h has been omitted due to the large value caused by the low OD600. Note: Fluorescence* is calculated as: (the measured fluorescence value) - (the fluorescence of LB medium).

As shown in Fig.6 (a), Mut9 had less leakage of expression during non-induction than Mut5 and Mut9. Fig.6 (b) also shows that this Mut9 is also induced to a level comparable to the original when arabinose is induced. Based on these results, we found that mutant 9 of Part:BBa_R0062 with reduced leakage is a more suitable lux promoter for our project.

Achievement of autonomous population regulation

We induced quorum sensing in E. coli and confirmed lysis occurring in response to an increase in the number of surrounding E. coli cells via OD600 measurement.

Fig.7 The OD600 graphs when luxI controlled by the araBAD promoter was induced with 0.2% arabinose and when it was not induced, for two cases: (a) using the lambda phage lysis cassette and (b) using ColE7.

As shown in Fig.7, we successfully induced lysis through quorum sensing for both types of genes. In the case of the lambda lysis cassette, bacterial growth stopped at OD600 ≈ 0.5, while in the case of ColE7, it stopped at OD600 ≈ 1. However, in both cases of lysis genes, non-lysing mutants started proliferating and couldn't be prevented from inhibiting the increase in OD after 10 hours. This emphasizes the need to prevent non-lysing mutants from dominating the culture. " Finally, based on the results and the experiment of sfGFP leakage described in Protein degradation, we have determined that among the two types of lysis genes used in this study, the lambda phage lysis cassette is more suitable for our project.

We designed a modified version of Part:BBa_K2020002 that includes a his6-tag for subtilisin, and induced the expression of subtilisin in E. coli by adding IPTG. This construct contains a specific signal sequence at the N-terminus, which is expected to facilitate secretion of the subtilisin into the extracellular space. The His6-tag remains attached to the recombinant protein even if the signal sequence is removed because it is located at the C-terminus of subtilisin. We cultured BL21(DE3) via expression induction, separated the culture into supernatant and pellet fractions, and then performed enzyme purification from each fraction.

For the supernatant, we directly mixed the cultured supernatant with nickel beads and attempted to retrieve the proteins that would bind to them. However, with this method, we could not recover an amount of recombinant protein exceeding the detection limit.

Considering the possibility that the expressed recombinant protein might be retained inside the cells, we subjected the pellet to ultrasonication to disrupt the cells. Subsequently, we attempted to recover subtilisin using nickel bead-based affinity chromatography from the centrifuged supernatant. Following this, we conducted a Pierce 660nm Protein Assay, and confirmed the presence of a protein consistent with subtilisin.

Fig.9 Intracellular protein concentration of purified protein from E. coli cells,

Concentrations were assessed using the Pierce 660nm Protein Assay.

Confirmation of subtilisin secretion into the extracellular space via Western blotting Subsequently, we conducted Western blotting using a mouse anti-His-tag monoclonal antibody (1:2000) purchased from MBL Life Science.

Fig.10 Results of Western blotting

From left to right: purified subtilisin (MW= 37.4 kDa), TCA-precipitated culture supernatant (with a slight amount of TCA residue), culture supernatant, E. coli pellet, supernatant obtained after ultrasonication and centrifugation of E. coli, precipitate obtained after ultrasonication and centrifugation of E. coli, and TCA-precipitated culture supernatant (TCA was completely washed).

As evident from Fig.10, samples concentrated from the E. coli culture supernatant (lanes 3 and 8) also yielded bands of similar length to subtilisin. This suggests that subtilisin was expressed and secreted into the extracellular space. However, when the culture supernatant was assayed without concentration, the bands were hardly detectable, implying that the quantity present in the medium was not significant.

Furthermore, from lanes 5-7, it is apparent that subtilisin is well-expressed within E. coli, but much of it is found in the pellet fraction. This suggests that secretion may have taken longer than expected.

From these results, it can be concluded that subtilisin was secreted into the extracellular space. Interestingly, similarly short bands were observed in both the purified protein and from inside the E. coli cells. These are presumed to be partial degradation products of subtilisin sharing the C-terminal His6-tag. Salamin et al. (2010) [19] reported in their paper that their subtilisin exhibited self-cleavage activity, and at higher concentrations, the full-length form became less prevalent. Although their protein is different from ours, we inferred that our subtilisin also exhibits similar activity, resulting in the observed partial degradation products.

We aimed to construct a system for recycling proteins derived from lysed E. coli using secreted subtilisin. However, it has not been experimentally demonstrated whether the disruption of the cell membrane by lysis would lead to sufficient leakage of internal soluble proteins into the medium.

To address this, we expressed sfGFP inside E. coli and induced lysis with our two lysis cassettes separately. The lysis cassettes from lambda phage and ColE7 were downstream of the araBAD promoter and were induced by arabinose. We measured the fluorescence intensity of sfGFP present in the supernatant obtained by centrifuging the culture after lysis and compared it to the case where arabinose induction did not occur. As a result, we confirmed that the induction of lysis, as shown in the graph below, led to significantly higher fluorescence, indicating increased leakage of sfGFP into the medium.

Fig.11 Comparison of Fluorescence* / OD600 of sfGFP released into the supernatant of arabinose-induced and uninduced lambda phage lysis cassettes and ColE7

(Fluorescence* = fluorescence measurement - LB medium fluorescence).

Based on the results above, we can conclude that lysis results in the release of E. coli's intracellular proteins into the culture medium. It is expected that other proteins, not just sfGFP, will be similarly released. Furthermore, as mentioned earlier, we have also established a mechanism for subtilisin secretion into the extracellular space. By combining these two systems, it becomes evident that it is realistically feasible to degrade proteins released from lysed E. coli and reuse them as a nutrient source.

The activity of nucA was unexpectedly revealed. When we conducted direct colony PCR for E. coli colonies expressing active nucA and those expressing inactive nucA, we obtained unclear bands only from the colonies expressing active nucA.

Fig.13 Electrophoresis results after conducting direct colony PCR on three separate colonies of E. coli expressing inactive nucA and three separate colonies expressing active nucA

Lanes 2-4 correspond to direct colony PCR of E. coli colonies expressing inactive nucA, while lanes 5-7 correspond to those of active nucA.

As shown in Fig.13, bands amplified from E. coli. expressing active nucA are clearly smeared (lanes 5-7). In addition to this photograph, we have observed multiple instances of abnormal bands believed to be caused by nucA.

We were intrigued by the abnormalities observed in direct colony PCR, so we purified plasmids from various colonies and confirmed their sequences. As a result, mutations causing the loss of nucA function were found in all samples from colonies that produced clear full-length bands. In contrast, plasmids obtained from the samples from colonies that produced smeared bands were found to encode the wild-type nucA sequence. This reveals a clear correlation between the PCR product electrophoresis pattern and nucA activity.

We did not fully understand the exact reasons for the changes in the PCR product electrophoresis pattern. Before the direct colony PCR, it is possible that nucA was activated, leading to cleavage of the plasmid due to its endonuclease activity, resulting in small fragments that could nonspecifically bind to the primers and create smeared signals during the PCR cycles.

We induced lysis in E. coli., then performed real-time PCR using the supernatant and quantified the amount of leaked DNA in the supernatant.

Fig.14

(a) Results of real-time PCR conducted on the supernatant of E. coli culture in which the lambda phage lysis cassette was induced at an arabinose concentration of 0.2% and cultured for 5 hours.
(b) Results from cells with both lambda phage lysis cassette and nucA induced.
(c) Results from cells with only nucA induced.

We conducted DNA quantification to confirm the leakage of DNA from E. coli into the culture medium after inducing lysis. Specifically, we amplified a 262bp sequence using real-time PCR, using a portion of the common plasmid sequence as a template for each strain. As shown in Fig.14(a), it was confirmed that a certain amount of plasmid was actually leaked into the culture medium after lysis. On the other hand, when we attempted to amplify the same sequence from the culture supernatant of E. coli that expressed nucA along with the lysis cassette, as shown in Fig.14(b), no amplification was observed. In the supernatant of cultures that were not induced for lysis and cultivated, plasmids were not observed, as shown in Fig.14(c). From these results, it was demonstrated that DNA from E. coli debris was released into the culture supernatant during lysis, and that this DNA could be efficiently degraded by expressing nucA in the periplasm of the same E. coli.

As expected, we were able to create and demonstrate the mechanism by which nucA degrades DNA during lysis. The degraded DNA is expected to be utilized as a nutrient source, allowing the next generation of E. coli to grow efficiently.

Fig.16 Conceptual diagram of our gene circuit

When arabinose induces the araBAD promoter-controlled BxbI integrase, BxbI integrase is expressed. By appropriately placing the recognition sequence of BxbI integrase within this gene circuit, it allows the reconstitution of the previously separated sfGFP sequence through recombination. At this point, there is a 15-amino acid scar that remains within the reconstituted sfGFP. However, when BxbI integrase expression was induced by arabinose, the fluorescence of this sfGFP was observed, indicating that this scar did not affect its function.

Fig.17 Comparison of Fluorescence* / OD600 values of three types of cultures subjected to overnight shaking at 37°C.

The three strains are where BxbI integrase was induced with 0.2% arabinose, when it was not induced, and the gene expressing the integrase was not present. (Fluorescence* = fluorescence measurement - LB medium fluorescence.)

To examine the plasmid sequences, we transformed E. coli using plasmids obtained from E. coli induced with arabinose. We then examined the sequences encoded in the plasmids retained by the fluorescent colonies among the colonies that grew.

From the above results, it can be concluded that by appropriately selecting the scar insertion site, the expression of the target protein dependent on DNA recombination by the integrase can be induced. This also can be applied to our project's target genes, LuxI and LuxR. By predicting a suitable location for scar insertion and implementing split genes, initiation of QS-Lysis through the integrase expression can be achieved. Information about these two factor locations is provided on Model.

Fig.18 Sanger sequencing of the plasmids carried by the fluorescent colonies

This result confirmed that the 15AA scar left behind by BxbI integrase recombination was present.