IPTG is associated with cell deconstruction and cytotoxicity. In order to sufficiently assess the impact of our inducer to cell density, we methodically examine a variety of IPTG concentrations and their effect providing valuable results to maintain cell viability.

The growth behavior of B.subtilis was determined by measuring cell absorbance at 600 nm (OD600). B.subtilis 3601 were inoculated in LB/BG-11 medium with IPTG concentrations at 0, 0.05, 0.1, 0.2, 0.4 mM. Ηourly OD readings were taken over 24 hours.

From the OD600 values we investigated the effect of IPTG variation in cell density on the stationary phase when it hits its maximum. The control culture (no IPTG in the media) showed, as expected, more exponential growth rate. Cultures with 0.05 and 0.1 mM IPTG in the media had a small deviation from the control, while for IPTG concentrations over 0.2mM the deviation was more distinct.

Our concerns about IPTG cytotoxicity were experimentally confirmed. Higher concentrations, although yields in greater induction according to the literature, also negatively affect cell density. We decided that the optimal IPTG concentration in line with the objectives of our project is up to 0.1mM. However, comparing recombinant hosts is considered crucial in order to safely conclude that at that concentration IPTG, the inducer is suitable without affecting cell growth and metabolism. Therefore more experiments about metabolic burden needed to be performed in recombinant cultures.

We took into account the measurements and results of cycle I for B.subtilis 3601. To achieve optimal induction we should consider the highest IPTG concentrations yet with the minimum, possible, negative effect of IPTG on cell growth. Therefore we purpose 0.05 and 0.1mM IPTG as optimal concentrations in order to get the best possible outcome. It is then vital to investigate how this concentration affects individual Nostoc growth, considering the future coexistence with B.subtilis within the consortium.

Nostoc's slow and multifactorial growth rate, did not allow us to investigate IPTG and other factors variations separately due to time limitations. Therefore the light density kept stably low simulating soil conditions. The growth behavior of Nostoc Oryzae TAU-MAC 2710 was once again determined by measuring cell absorbance at 600nm (OD600). Two cultures were inoculated in LB/BG-11 medium with IPTG concentration at 0.1 mM (tested culture) and in IPTG absence (control culture).

OD measurements were taken on a daily basis in order to test IPTG optimal concentration and culture conditions. Test culture has grown very slowly over 20 days when Nostoc is still in its exponential phase. The growth curve of tested culture exhibited slowed down progression of the exponential phase. We suspected that either IPTG affected cell growth or the UV intensity could also be considered as a decelerating factor.

Both IPTG and low light conditions were necessary for the success of Euphoresis. Hence we decided to further test the culture in such harsh light conditions in order to be able to thrive under soil as well as the second best IPTG concentration of IPTG at 0.05mM. Furthermore, since we already knew about Nostoc’s slow growth rates in such cases, the team decided to implement an alternative kill switch circuit. In such design we coupled the circuit with a quorum sensing mechanism so that Nostoc’s kill switch would be induced by the signaling molecule that will be produced by B.subtilis (exhibiting high growth rate) while its expression will be still IPTG inducible.

In the redesign kill switch mechanisms we coupled the LuxR/LuxI quorum sensing. Particularly, we propose that B. subtilis will contain the TetR gene controlled by IPTG inducible promoter and when the inducer is present in the media TetR gene is activated contributing to cell survival while in absence cell death. Accordingly LuxI activation will produce AHL molecules that when diffused into Nostoc cells contributes to cell survival, while in absence of IPTG cell death. Cell survival in Nostoc will be achieved by expression of Antitoxin (VapB15) that forms clusters with constitutively expressed toxin (VapC15) and deactivates it. While for B. subtilis cell survival is achieved with TetR expression that represses BsrG toxin production. Once the improved kill switch design was developed and the optimal strain from the respective engineering cycle was selected, recombinant cultures of B. subtilis WB800 needed to be tested in order to examine the metabolic burden of recombinant hosts due to IPTG induction in the strain we selected. Particularly when combined with recombinant hosts, IPTG - induction affects the expressing conditions and adds further metabolic cost. Providing the least possible metabolic burden while ensuring biosafety, is crucial for sufficient expression of Laccases in B. subtilis and HetR in Nostoc in order to achieve phenolic compounds degradation and nitrogen fixations in respect with biosafety, that is Euphoresis goal.

To investigate the additional metabolic burden on B. subtilis due to IPTG induction two cultures were examined. B. subtilis 3601 (not recombinant) from the first OD600 measurements and B. subtilis WB800 pJUMP29-1D’ (tested). Hourly OD readings were taken over 24 hours in a similar manner. Unfortunately, Nostoc’s demanding transformation conditions did not allow us to investigate the additional cost of metabolism in IPTG presence because of time limitations. However the second purposed IPTG concentration was tested in a non recombinant host to examine IPTG impact on growth. (Later we confirmed HetR production with an SDS page in those concentrations. See results )

B.subtilis tested cultures were inoculated in LB/BG-11 medium with IPTG concentration at 0, 0.05, 0.1, 0.2 and 0.4 mM while Nostoc cultures were grown also in LB/BG-11 medium enriched with 0.05mM of IPTG and low light conditions. To obtain results that can be properly evaluated according to the first measurements, B.subtilis hourly OD readings were performed over 24 hours and Nostoc daily OD measurements were conducted over 20 days.

As expected, B.subtilis pJUMP29-1D’-WB800 exhibits overall lower OD values compared to 3601 strain, but surprisingly with small deviation. pJUMP29-1D’-WB800 showed significant deviation between up to 0.1mM IPTG, whose growth rates were similar to those without and over 0.2mM that the reduced cell density is noticeable. According to the results for Nostoc Oryzae TAU-MAC 2710, at 0.05 mM no significant deviation was observed compared to the IPTG concentration of 0.1 mM. Thus it is concluded that we can test in our further experiments IPTG inducer concentrations of 0.05 and 0.1 mM for the two microorganisms to evaluate effectiveness of the improved kill switch design and the genes of interest (laccases and HetR), while in the future more experiments need to be performed on recombinant Nostoc Oryzae TAU-MAC 2710.

Our goal was our Bacillus subtilis to produce extracellular laccases. In order to ensure that our cells were producing laccases we had to verify their extracellular expression. Although lacasses are endogenous proteins naturally produced in B.subtilis, we had to ensure their exogenous production, as well as to test their activity. Thus we designed the sequence of our peptide with a TAT sequence that signals their extracellular transportation.

We selected the Bacillus 3601 strain that was available in our laboratory. Therefore we designed laccase’s gene sequence along with TAT signaling sequence. First we decided to follow Biobrick assembly because of the technique’s reliability, compatibility with iGEM characterization and flexibility in future designs. We performed both Biobrick assembly and the assembly with Mr Giannakopoulos’ protocol due to unexpected results in other assemblies. (see Optimal Promoters Engineering). Next we consulted our instructor and utilized his protocol in order to overcome the obstacles of the failed assembly. Then we selected restriction enzymes that align with our digestion (and do not destroy our gene of interest) and thus utilized E.coRI and PstI enzymes. Next, following the protocol a mastermix was prepared using a ligation buffer along with an enzymes-buffer ensuring that restriction enzymes will have the optimal conditions to be activated at their maximum. Digestion and ligation performed in one run and the cultures incubated in 37°C. We confirmed an indication of assembly by PCR primers. Then we ran gel electrophoresis and interpreted the corresponding bands. If we get the expected bands then SDS page and Bradford is crucial to be performed in order to define quantitatively endogenous and exogenous protein production respectively.

Agarose gel’s results were interpreted and successful assembly was achieved. Therefore we proceed to SDS and Bradford experiments as planned. The two techniques will provide us more accurate results reducing error margin. Knowing the molecular weight of our laccase we were able to qualitatively examine its expression by comparing gel bands between the control and tested samples. We expect the line of the molecular weight that corresponds to lacasse’s molecular weight in the SDS page to be enhanced compared to the control one. Additionally we expect an increase of total protein in Bradford. Four cultures of B.subtilis 3610, that was available in our lab, were inoculated with 0, 50, 75 and 100 μΜ CuSO₄. Four samples from each culture were examined on SDS page and Bradford.

According to the results of the gel bands provided, we observed no indication of lacasse endogenous or exogenous protein. Surprisingly, we could not understand why, since the assembly was tested and successful. Hence we started to investigate possible lab errors and reach out for scientific feedback.

After contacting PhD candidate in Biotechnology and Synthetic Biology Anargyros Alexiou we realized that the problem was the proteases that our strain produces, which destroy our laccase and that is the reason that our laccase was not detected in the extracellular matrix. Following that valuable insight, we decided to choose the strain of Bacillus subtilis WB800, originating from Bacillus subtilis 168 strain with 7 genes of extracellular proteases being knocked out. The extracellular proteases of B. subtilis possibly destroyed our laccase.

Experimental design was in line with the previous one since the exact same procedures will be tested in a different strain. So we transformed B.subtilis WB800 with the lacasse recombinant gene and conducted Bradford and SDS pages to quantitatively investigate protein expression. We also decided to test different concentrations of CuSO₄, since we were informed that it is crucial and increases lacasse activity and production.

In four cultures of B.subtilis WB800 with concentrations of 0, 50, 75 and 100 μL CuSO₄ Bradford and SDS page were conducted. We expected a higher band density around 77kDa for SDS page as the concentration CuSO₄ is rising, while in Bradford increased extracellular total exogenous protein in respect with the rising of CuSO₄.

According to the results from Bradford, with CuSO4 rise, the protein production rises as well. CuSO₄ is related with enzyme’s function. In SDS page results, there is a notable protein concentration increase around 75kDa, which is around laccasse’s molecular weight. With those two indications we can assume, a successful laccasse production.

According to the data derived from Effects of IPTG engineering, two values of IPTG concentration are proposed in order to serve Euphoresis objectives. The improved kill switch containing the activation genes in B.subtilis was designed and needed to be tested in the particular values of IPTG. Unfortunately as the research continues, we decided that maybe it should be more suitable to also use an IPTG inducible promoter for LuxI activation, in order to be able to control both kill switches under the same factor. Therefore, we redesigned Nostoc’s kill switch and placed a second order. First we needed to follow carefully the techniques that best describe our objectives before reaching the final step, that is IPTG induction.

Effectiveness of the circuit activation genes needed to be achieved performing the suitable assembly. In this stage of the project, we chose to perform Biobrick Assembly since it is a standard and easy assembly method that serves educational purposes. Kill switch demonstration was in the initial phase and we had already the first misguided choice, of choosing non IPTG inducible promoter (pGroE) when there was a better option (P_grac). Therefore utilizing Biobrick Assembly we ensure that if further adjustments need to be performed in the future and the genetic circuit to be more complex, with Biobrick we save precious time because of the method’s universal prefix and suffix.

We used E.coRI and PstI restriction enzymes, after making sure that they didn’t cut somewhere between the prefix and suffix where the genes of interest are located. Afterwards we will demonstrate double digestion and ligation. The cultures have grown successfully and PCR amplification followed. Gel electrophoresis was followed next, in order to verify the molecular weights of each expected fraction by the restriction enzyme according to the ladder. Then we may have sufficiently validated results. Gel was not successful and no bands presented.

Although Biobrick has some advantages that we wanted to exploit, it comes with certain disadvantages that we had already been briefed from iGEM. Especially about the method’s yield of success. We suspected that the method's overall yield was the problem, since no band was observed.

Taking into consideration the results from the previous failed assembly and iGEM’s notification about the method’s yield we needed to examine if the composite parts assembly was successful before proceeding to further investigations.

Driven by our instructor’s advice Mr Giannakopoulos, we utilize an original protocol made by himself in order to overcome such barriers. According to that protocol, an additional buffer is included in the mastermix that ensures that restriction enzymes will have the optimal conditions to be activated at their maximum. The additional presence of the enzyme buffer along with the ligase buffer is one of their major differences. Furthermore, when utilizing the thermocycler for the assembly reaction mixture, the three distinguished steps contribute to background digestion.

Similarly, our aim was to first verify a successful assembly before moving to further investigations regarding gene expression and inducer variation. In order to achieve that, we selected EcorI and PstI , performed digestion and ligation in one run according to Mr. Giannakopoulos’ protocol. After cultures’ successful growth, we performed PCR quantification and ran agarose gel, screen fractions’ molecular weights and interpreted the data.

According to the results, we achieved a successful assembly and agarose gel’s bands correspond to the expected MW of the fractions digested with the restriction enzymes. We suggest other iGEM teams facing similar obstacles with the BioBrick assembly to use it (see Contribution).

We therefore decided to follow Mr Giannakopoulos protocol to assemble the composite part containing the LuxI gene under control of P_grac promoter, that arrived belatedly. Unfortunately, although the assembly was achieved, we were not able to perform experiments designed for quantification of fluorescence. On the other hand, the assembly composite part of the ptRc-02 derivative promoter controlling TetR expression was successfully completed and needed to be investigated in regard to efficient BsrG repression due to the TetR repressor.

We therefore designed an SDS page in order to be able to determine the variation of IPTG effect on BsrG expression. Additionally, we investigated in silico the effect of IPTG induction under different concentrations on promoter induction. More specifically the production of proteins under control of IPTG inducible promoters was tested in different circumstances: with varying IPTG concentration, inducer association constant and plasmid copy number. At the same time, according to in silico analysis, when p_GraC and pTrC02 derived promoters are used to control LuxI and TetR production respectively, there are a few ways to accomplish coordinated induction. Those variables are about plasmid copy number and promoter association constant. However in both silico analysis about BsrG (toxin) and VapB15 (nostoc antitoxin) under different copy numbers, they achieved desirable outcomes utilizing medium vectors.

According to the data, one band had no correlation with IPTG when supposed to and control and tested cultures did not show the expected bands according to their molecular weight.

According to the data, we observed that BsrG toxin production in B.subtilis is not regulated effectively by IPTG. Therefore, we conclude that utilizing the same IPTG inducible promoter P_grac to both genes LuxI and TetR that trigger the kill switch, could be proved suitable, since our vector is suitable.

The improved design contained the LuxI gene under control of an IPTG inducible promoter p_GraC and the TetR gene under control of another IPTG inducible pGraC promoter. We constructed reporter plasmids in order to include process controls, and fluorescent proteins at each step of the kill switch in order to efficiently quantify TetR, BsrG for B.subtilis and LuxI, LuxR, Antitoxin production for Nostoc. However due to time limitations, quantification of the kill switches mechanisms could not be completed and need to be investigated in the future.

In our design we investigated both pectin and chitosan properties regarding the desired characteristics of the final product. Pectin provides high swelling ability, while chitosan enhances stability. After consulting with Professor Peppas, we chose the ratio of CS2%PE1%, CS3%PE1% so that we will be able to exploit both polymers characteristics, while avoiding the risk of pectin’s instability when it is in excess.

When a new material is designed, its desired properties should be followed by its corresponding physicochemical properties in order for the characterisation to be accurate and valid. Hence viscosity measurement tests were crucial to conduct first, along with swelling tests. This investigation will provide us a comprehensive overview of the crosslink network for each proposed ratio.

Since at pH = 7 pectin’s charge is negative, while chitosan’s charge is positive, the two polymers crosslink was conducted by addition of fully dissolved pectin in chitosan at 50°C in solution of 0.1 M hydrochloric acid. 0.5 mL of pure acetic acid was then added in each beaker and left under stirring for 24h. Viscosity was determined utilizing an electronic rheometer at temperatures 25°C and 37°C, 5 days after the formation. After the samples were dried for 2-3 days, swelling tests were performed in time = 0, 10, 20, 30, 60, 120 minutes and 24 hours by measuring initial dry mass of each sample and the dry mass of tested samples after swelling.

Swelling ability of CS2%PE1%, CS3%PE1% exhibits similar values. In pH 7.3 they have already reached swelling equilibrium below 200 minutes and by 1400 minutes they still present swelling. While for pH 5 they seem not to have reached swelling equilibrium at 1400 minutes, indicating a much higher swelling capacity at those conditions.

The choice of the correct ratio in our hydrogel was dependent on the degradation rate/stability of our samples. Therefore we needed to test all hydrogel samples in a soil environment within 20 days. Degradation of the hydrogel matrix is highly affected by stability in soil, so such an examination is considered vital for our desired goal in order to evaluate hydrogel properties based on their lifetime.

In order to test the degradation rate/stability of our hydrogel was tested in soil. The dried sample of the hydrogel was placed inside the soil. It was weighed at the start and then every 7 days for a month to predict its lifespan. The sample were at room temperature

Soil’s pH was measured at pH=6.85 , a value within the range of greek forests’ soil, as said by Prof. Tsantilas. By tracking each sample's mass loss, the produced hydrogels' soil stability was assessed. Each dry scaffold's part was carefully divided and then put into a plastic cup that had been partially filled with soil samples. The soil was then added to the cup until it was full.The samples' weight was measured once more after each interval and stability/degradation was determined.

According to the data, we can fairly conclude that the CS2%PE1% hydrogel can absorb external moisture and hasn't degraded throughout the course of the 20 days of the studies due to no mass loss. These measurements lead us to choose CS2%PE1% hydrogel as the best material for our project.