3.07.2023

Our scientific team held an important meeting to delve into the basic methods of synthetic biology and elaborate our topic. During the meeting, we analyzed the latest developments in the field of synthetic biology and explored the possibilities that this approach offers to solve our research topic. Our discussions focused on topics such as gene design, genetic modification of organisms. This meeting is an important step towards understanding and utilizing the modern tools of synthetic biology to solve our scientific research challenges.

5.07.2023

Our scientific team had a second meeting to determine the techniques to be applied and to find the protocols that will be used in the future. In this meeting, we extensively reviewed the available literature and previous research in our field in order to select the most suitable techniques for achieving our objectives. Subsequently, we discussed and defined the protocols that will be followed for the execution of our research activities, taking into consideration our expectations for accuracy and reliability in our results. This meeting represents a significant step towards the successful fulfillment of our research goals and the advancement of our scientific progress.

6.07.2023

Our scientific team conducted an extensive research aiming to locate marbles that had been exposed to the environment for decades, subjected to colonization by numerous microorganisms. Our analysis included identifying the composition and structure of these marbles, as well as examining the effects that microorganisms had on their surface and quality. The results of our study reveal the intricate process of biological impact on the stony core of the marbles and offer significant insights into understanding the mechanisms of colonization and alteration of mineral materials by the biological factor. This research highlights the importance of interdisciplinary collaboration in the fields of geology and biology for exploring complex interactions between the geological and biological worlds.

7.07.2023

Our scientific team successfully carried out the process of isolating microorganisms from the marbles of the area of Patras, Greece. The methodology involved continuous serial dilutions of the samples, as well as the creation of nutrient material designed for the growth of bacteria and fungi, which are found in the literature in the marbles of the area. This methodology allowed us to reveal the diversity of microorganisms present in these marbles. Our analyzes will contribute to the understanding of the biological diversity in the environment of the area and to further research on the interaction of microorganisms with the environment and marble materials but also in the future to the production of antimicrobial peptides where we aim to realize if these peptides eliminate the these microorganisms.

15.8.2023

Order of the insert DNA sequences from IDT

29.08.2023

Sterilization.

Preparation of a stock solution of chloramphenicol 167 mg/ml in ethanol.

Preparation of 100 ml liquid LB and 100 ml solid, addition of chloramphenicol to a final concentration of C = 50 μg/ml, and spreading it on 5 petri dishes.

30.08.2023

Transformation of E. coli with the plasmid ps61c3 from plates K16 and O16.

31.08.2023

Preparation of wet cultures from single colonies of transformed E. coli in 20ml LB in colonies.

Storing of the pedri dishes in the fridge.

01.09.2023

Isolation of the plasmid with a final concentration of C=6 ng/ml, which was very low.

The antibiotic concentration may be correct, and for an additional check, we will culture non-transformed E. coli on a petri dish with the antibiotic. If we do not observe colonies even up to 24 hours later, we will isolate the plasmid again.

04.09.2023

We conducted a culture of E. coli on a petri dish with antibiotics to check if it is fine.

05.09.2023

The E. coli did not grow, so we conclude that we have transformed ones in the fridge. We cultured single colonies on solid media. We also set up two liquid cultures, one from each.

06.09.2023

Storage of cultures in the refrigerator (+ 4℃).

Dilution of primers

PCR for :

- PSB1C3 K16 from the plate plasmid with primers for the plasmid

- An insert DNA sequence with primers for the insert

- PSB1C3 Plasmid K16 from the plate with primers for its insert DNA sequence

- Single colony from the transformed E.Coli with PSB1C3 K16 with primers its insert DNA.

- Single colony from the transformed E.Coli with PSB1C3 K16 with primers for the plasmid.

Electrophoresis on a 2% agarose gel at 120V with a ladder as a 1000 b.p. marker.

The PCR reaction turned out to be infected.

We prepared 200ml of liquid nutrient and 200ml of LB with chloramphenicol.

We sterilized the materials and nutrients, plated them on solid media, and then stored them in the refrigerator (+ 4℃).

07.09.2023

The same PCR as on 06.09.23, was repeated, and it came out without infection, where it was revealed by electrophoresis on a 0.8-1% agarose gel at 120V.

The gel was very fragile; it's better to use 1.5-2% agarose gel.

18.09.2023

PCR For all inserts

The gel was very fragile; it's better to use 1.5-2% agarose gel.

19.09.2023

REPEAT PCR1

We prepared 200ml of liquid nutrient and 200ml of LB without antibiotic DH5a 🡪 on petri dish

22.09.2023

Electrophoresis of th PCR products at 1% agarose gel.

Gel extraxion of the desired bands.

Dilusion of the PCR products.

Photometry of the Samples.

Dilution of the PCR products.

23.09.2023

Ligation of the PCR products with PSB1C3 plasmid.

24.09.2023

Transformation of competent DH5A E.Coli cells with the half of the ligation products.

27.09.2023

The transformation was successful, so we stored the pedri dishes in the fridge and performed transformation for the rest of the ligation products.

28.09.2023

In order to detect if the colonies express our insert sequences, we chose two specific dishes, one with transformed E.Coli with sfGFP protein and Tachyplesin and one (cintrol) with GFP and no Tachyplesin, and we lifgted them with UV, to see if they flourescence. We observed that none of the dishes had fluorescence colonies.

Storage of the pedri dishes in the fridge.

2.10.2023

After a lot of bibliography research, we found out that DH5A E.Coli cells are not able to express proteins, because of not having the T7 RNA polymerase. Our insertsjad T7 promoter, so the transcription could not start. We decided to repeat our experiments with BL-21 (DE3) E.Coli cells, which have T7 RNA polymerase and are appropriate for protein expression.

3.10.2023

PCR for:

- PSB1C3.

- Electrophoresis.

- Gel extraction.

4.10.2023

Dilusion of the PCR product.

Ligation of half inserts with PSB1C3.

Preparation of competent BL21(DE3) competent cells.

Transformation.

6.10.2023

We did not observe colonies. Transformation was unsuccessful. Being sure that PCR, ligations and the transformation protocol were no problem, we thought that the cells were not competent. So, we prepared new buffer and made competent BL21(DE3) again.

We transformed cells with two ligations at this time, in order not to spend all of the remaining ligation products.

7.10.2023

Transformation was unsuccessful again. We thought that maybe the BL-21(DE3) grow so fast, that in the first 3 hours of growth they overcome the start of the exponential stage of growth, when they are able to become competent with the buffer. So, we photometered them at the first 3 hours, when they are supposed to be ready and realised that their OD=1. We wanted it to be about 0.4. So we started culturing cells again and photometered them every 20 minutes until they have OD=0.4.

9.10.2023

So we started culturing cells again and photometered them every 20 minutes until they have OD=0.4. It took about an hour. We don’t know the reason why these BL-21(DE3) were growing so fast.

10.10.23

We transformed the cells with the remaining ligation products.

11.10.23

The transformation was successful! We managed to have E.Coli cells producing Tachyplsin, but we have not time to appreciate its antimicrobial action.