Methods:

Isopropyl β-D-1-thiogalactopyranoside (IPTG) is a molecular biology reagent, a mimic of allolactose (lactose metabolite). IPTG controls the expression of the promoters related to the quorum sensing system, and the kill switches. However, IPTG in high concentrations has toxic action in cells. So, we had to test the effect of different IPTG concentrations in the bacteria population. At the start of our experiments, we conducted the assemblies using the strain Bacillus subtilis 3601. Before the parts for the assemblies arrived, we had to check the IPTG variation effect. The cultures were grown ON at 30°C, in BG/LB medium. The next day, we conducted spectrophotometry OD600 measurements after inducing them with 0.05mM, 0.1mM, 0.2mM, 0.4mM, and one control sample with no IPTG. The collection of the data was done every hour for 24h.

Results:

 Conclution:

After examining the growth rates we realized that IPTG in concentration higher than 0.1 mM has a negative effect on B. subtilis growth. The following experiments for the growth rates of Nostoc were conducted in the range of 0-0.1

Methods:

Originally, our plan was to design the kill switch of the cyanobacterium in a similar way with the B. subtilis. More specifically, we wanted to have the same induction system: an IPTG induced system. We decided on the use strain of Nostoc Oryzae TAU-MAC 2710 with the guidance of our PI Prof. Gkelis. After choosing the strain we had to test its growth rates on different IPTG concentrations. Having found out that concentrations more than 0,1 mM is toxic for B. subtilis, we tested the cyanobacteria cultures in 0.05 mM, and 0.1 mM and one control with no IPTG. The cultures were grown in 30oC, in LB/BG medium. Because the cyanobacterium metabolic rhythm is affected by the light conditions, we made sure that our cultures were in limited light to simulate the conditions in the first layer of the soil. We measure our samples at 680, 750, and 800 nm, every two days.

Results:

Methods:

For the conduction of our experiments we needed a plasmid backbone with medium copy number. According to our model results a plasmid with medium copy number is proposed to satisfy at the same time the needs for our antitoxin production, the coordination of TetR and LuxI, and the production of the Bacillus toxin. We decided to use the plasmids with medium copy numbers from the iGEM Distribution Kit. We transformed TOP10 E.Coli cells with the plasmids I8, C12, E12, K8, M8, E9, G8, and A12 and then the cells were plated and incubated at 37oC, overnight. Every plasmid contained a resistant gene for the antibiotic of kanamycin so, in order to detect the transformed E.coli cells, the plating was done in LB agar with kanamycin. Only the cells that have received the plasmid survived and formed colonies in the LB Agar. The transformation of E.Coli cells was successful for all the eight plasmids.

We inoculated into liquid growth media one culture of each plate and we grew the cells at 37°C overnight. Then we proceeded to plasmid extraction followed by NanoDrop spectrometry.

NanoDrop spectrometry is used to determine the purity of the plasmids.

All nucleotides absorb at 260 nm, meaning that absorbance in a different wavelength may indicate the presence of other molecules, like a protein. The 260 nm/280 nm ratio can be used as a primary indicator of the purity of DNA. A ratio of ~1.8 is indicative for “pure” DNA. The 260/230 ratio is a secondary indicator of DNA purity and indicates “pure” DNA in the 2.0-2.2 rates.

Results:

From the NanoDrop spectrometry, the following results were obtained (C equals plasmid concentration in ng/μL):

 Conclution:

In order to conduct the assembly we needed an efficient plasmid concentration. All of the plasmid concentrations seemed to be satisfying, apart from the E9-E9’ concentration, which was below 20,000 ng/μL. However, 260/280 and 260/230 ratios indicated impurities in all the plasmids. Taking all those into account, we chose to use the C12 plasmid for our parts assembly, which seemed to have the best combination of 260/280 and 260/230 ratios, as well as a concentration that is considered satisfactory.

Methods:

We designed our sequences, with the respective prefix and suffix, to be compatible with the requirements of BioBrick assembly. At our first attempt, we tried to assemble our parts following the protocol of New England Biolab for the double digestion with restriction enzymes. We used the restriction enzymes E.coRI and PstI. After the plasmid was digested, we continued with the ligation according to the T4 DNA Ligase NEB protocol. After the two protocols, we used the recombinant plasmids to transform E. coli TOP 10 cells. The cultures of the cells were an initial proof of the successful transformation.

We designed the suitable primers for every desirable part and continued with the PCR procedure to amplify our genes of interest. We continued with the electrophoresis gel to validate their transformation.

Results:

Despite what we anticipated, the results of the gel electrophoresis showed that the transformation was not successful for the assembly with multiple fragments.

 Conclution:

We decided to try to modify the protocol for the assembly, using the same restriction enzymes to achieve assembly with multiple fragments.

Methods:

Knowing from the iGEM protocols that BioBrick assembly does not have high succession rate, and by following the consultation of our team’s instructor Christos Giannakopoulos we decided to follow a modified assembly protocol. More specifically we used a specific Golden Gate Assembly Protocol and instead of BsaI we added the restriction enzymes of BioBrick Assembly. At this point of our project the design for the promoter of LuxI changed because we wanted the immediate induction of cyanobacteria to be also IPTG induced. So we only recombined the composite parts regarding the Bacillus subtilis kill switch, laccase gene, and HetR gene. More specifically for our kill-switch we did an assembly of three composite parts, while for the other two it was one composite part assembly. The plasmid mixture was used to transform competent E. coli Top10 cells. Then we used the primers that we designed and we proceeded to the PCR procedure to amplify the desired genes. We continued with the electrophoresis gel to validate their transformation.

Results:

 Conclution:

The assembly was successful. The impressive part was that we managed to have a successful triple assembly for our kill-switch mechanism in one recombinant plasmid with our new protocol!

Methods:

We followed the protocol of minipreps and nanodrops from our successfully transformed with the desired recombinant E. colicells for B. subtilis 3601 transformation of the composite parts of laccase and Bacillus kill switch mechanism. After the transformation we prepared plate cultures in LB agar broth medium with kanamycin.

Results:

The transformation was successful, as we managed to have cultures grow in the plates

 Conclution:

Methods:

After the successful transformation of our B. subtilis cells we inoculate cultures from the plates with the transformed B. subtilis into a liquid growth medium. We incubate them in 37°C for 48h for the laccase production with 3 different concentration of CuSO₄ at 50 μΜ, 75 μΜ, 100μΜ, one transformed B.subtilis with 0 mM sample CuSO₄ and one control non transformed B. subtilis. After those 48h we centrifuge our samples and took 2 ml of the supernatant. First we calibrated with standard concentrations and then we calculated the concentration of our samples.

Results:

The resulting concentrations of our samples compared with the controlled ones were approximately the same. Although we conducted an SDS page in case we could detect a higher OD in the band of the molecular weight of our laccase in our samples compared to the controlled ones.

 Conclution:

Although the results of Bradford were not showing any enhanced protein production and extraction, we decided to conduct an SDS-page to see if there would be any significant changes in the expression of proteins in the molecular weight of our laccase.

Methods:

Following the protocol for the SDS page we used the same supernatant from the same samples as above in Bradford (3 different concentration of CuSO₄ at 50 μΜ, 75 μΜ, 100μΜ, one transformed B.subtilis 0 mM sample CuSO₄ and one control non transformed B. subtilis). Then we scan the resulted gel.

Results:

The protein volume in the band of the molecular weight of our laccase compared to the control sample had no significant difference.

 Conclution:

Because the recombination and transformation were successful we had to troubleshoot on what could interfere with the successful expression of our laccase. After contacting PhD candidate Anargyros Alexiou we realized that the problem was the extracellular protease that our strain was producing. He suggested the B. subtilis WB800N that has its extracellular proteases knocked out

Methods:

After changing the strain of B. subtilis we decided to calculate the growth rate after we transformed the B. subtilis WB800N to have more accurate results about its toxicity (because the extra plasmid increases the metabolic burden). We transformed our strain with pJUMP29-1D’ plasmid. Again, the cultures were grown ON at 30°C. The next day, we conducted spectrophotometry OD600 measurements after inducing them with 0.05mM, 0.1mM, 0.2mM, 0.4mM and control with no IPTG. The collection of the data was done every hour for 24h.

Results:

According to the results, we observe that cell density with IPTG inducer concentrations up to 0.1mM is similar to the control culture. On the contrary, cultures with IPTG above 0.2mM exhibit low cell density indicating slow metabolism and potential cytotoxicity.

 Conclution:

The growth rates of B. subtilis WB800N present decrease in cell density in IPTG concentrations higher than 0,1 mM.

Methods:

After changing the strain and testing the growth rates, we proceeded to transform this strain with the composite parts of laccase and the kill switch mechanism. We followed the protocol of minipreps and nanodrops from our successfully transformed E. coli cells with the desired recombinant plasmids for B. subtilis 3601 transformation of the composite parts of laccase and Bacillus kill switch mechanism. After the transformation we prepared plate cultures in LB agar broth medium with kanamycin.

Results:

The transformation was successful, as we managed to have cultures grow in the plates

 Conclution:

We started the characterization of our parts.

Methods:

After the successful transformation of our B. subtilis cells we inoculate cultures from the plates with the transformed B. subtilis into a liquid growth medium. We incubate them in 37°C for 48h for the laccase production with 3 different concentration of CuSO₄ at 50 μΜ, 75 μΜ, 100μΜ, one transformed B.subtilis with 0 mM sample CuSO₄ and one control non transformed B. subtilis. After those 48h we centrifuge our samples and took 2 ml of the supernatant. First we calibrated with standard concentrations and then we calculated the concentration of our samples.

Results:

The resulting concentrations of our samples compared with the controlled ones had differences compared with the controlled ones

 Conclution:

As it is proved, the raise of CuSO₄ concentrations cause a protein production rise. Since CuSO₄ is related to the copper functioning laccase enzyme, it can be implied that a rise of laccase production is detected when this molecule is present in higher concentrations.

Methods:

Following the protocol for the SDS page we used the same supernatant from the same samples as above in Bradford (3 different concentration of CuSO₄ at 50 μΜ, 75 μΜ, 100μΜ, one transformed B.subtilis with 0 mM sample CuSO₄ and one control non transformed B. subtilis). Then we scan the resulting gel.

Results:

The protein volume in the band of the molecular weight of our laccase compared to the control sample had significant differences

 Conclution:

A notable protein concentration raise, in the range of 75 kDa, was detected compared to the controls, implying a boosted laccase production as CuSO₄ concentration rises. Thus the production of our laccase can be considered successful an has indications that correlates with CuSO₄ concentration

Methods:

After the transformation of B. subtilis WB800N we inoculated into liquid growth medium with different concentrations of IPTG. More specifically we had liquid cultures 0 mM, 0,05 mM, 0,1 mM and one control non modified B. subtilis WB800N culture. We incubated the cultures for 24h at 37°C. We calculated the absorbancy in the band of the molecular weight of the toxin BsrG.

Results:

 Conclution:

There was no immediate correlation between the concentrations of IPTG and Our kill switch system. The desired result would be to have lower protein volume as the IPTG concentration increases. The results do not prove that. So, we decided to change the promoter of TetR with P_grac, as it was derived from a B. subtilis endogenous promoter and could have better results.

Methods:

Nostoc oryzae TAU-MAC 2710 was used for electrotransformation. The cells were selected in the log growth phase because the DNA can easily integrate foreign genes. We conducted plasmid extraction following the protocol of minipreps and nanodrops in order to extract the successful recombinant plasmids containing HetR composite part from E.coli transformation. After the transformation via electroporation and the inoculation of the cells into liquid growth medium with kanamycin, we conducted PCR and electrophoresis to prove the transformation.

Results:

The transformation of the Nostoc oryzae TAU-MAC 2710 was unsuccessful, as we did not see the fragment of our gene in the electrophoresis.

 Conclution:

After multiple failures in our tries to transform the cyanobacterium we decided to just try and see if the HetR protein is expressed in the E. coli cells

Methods:

After the failure to transform the cyanobacterium with our recombinant plasmid we decided to detect if the protein of HetR was being expressed in the competent E. coli cells. We centrifuge our sample and keep the cell pellet. We proceeded to cell lysis and then followed the protocol for the SDS page. We had samples from two liquid cultures with successfully transformed E. coli in LB medium with kanamycin and one control non transformed E. coli.

Results:

The results were showing higher protein volume in the band of the molecular weight of HetR in our samples than in our control.

 Conclution:

There is a possibility, because of the notable difference between the absorbance in samples and control that the promoter of HetR is functional as a promoter in E. coli, too. So, it is possible that the HetR protein was being produced.