Hardware

We based our hardware design on the hardware of the iGEM Thessaloniki 2019. It is an electrochemical DNA sensor, which has been used as quality control for our experiments. We kept the basic parts the same as the hardware of the iGEM Thessaloniki 2019 team, but we added some new elements, improving it. We added the insulation at a small part of the electrodes on the sensing board, in order to improve measurements considering the drift. We also added a temperature control system. We hope this electrochemical device to encourage iGEM teams to make precise and cost-effective instruments for their experiments. You can see in detail how we tried to improve the drift and temperature control on our hardware page, to implement our adjustment to your sensing boards, too

In order to help, we also provide a photo of the circuit of our electrochemical DNA sensor, if you want to preform this experiments and a protocol for electrode cleaning and sample immobilization in our protocol page.

Figure 1: Circuit of our electrochemical DNA sensor.

Troubleshooting

This is a guide for all iGEM teams having problems with transformation and competent cells. We hope we can help you!

During our experiments for the iGEM Interlab study of 2022, we encountered several unforeseen difficulties with bacterial transformation. We started our experiments by using E.coli (Beta 10, Neb) cells, the only available in our lab at that time. Unfortunately, in our first attempt to transform them with the green and red mRFP1 device plasmids from experiment 1 (BBa_J428112 and BBa_J428110) using Neb’s protocol for Beta 10 cells, there were no colonies in the agar plates.

In the beginning, we decided to change some of the conditions of the transformation, such as the amount of DNA that was used, the heat shock time, etc. All of our attempts were unsuccessful.

Table 1: Results of bacterial transformation in various transformation conditions

Afterward, we thought about the competence of our cells. We chose a protocol to make our cells more competent to plasmid DNA. This protocol was provided by iGEM Crete, within the framework of our partnership, to help us with the transformation of the cells. The protocol is submitted above to help future iGEM teams with their incompetent cells.

When the beta 10 cells were finally competent, we repeated the neb transformation protocol for Beta 10 cells, adding some different transformation conditions. The results are represented in the following table.

Table 2: Results of bacterial transformation after competence protocol in various transformation conditions

As you can see, the transformation was successful in all of our experiments after the application of the competent protocol. We learned that we should always check all of the factors of the experiments that we are doing and had a great time troubleshooting for a solution. To help future iGEM teams, we submit all of our results, including the competent bacteria protocol of iGEM Crete.

If you have any problems with your transformation protocols or you want more advice, we are no experts but we would love to answer all your questions. Feel free to contact us!