Proof of concept: Chip-experiments
Parallel to the experiments described above, we wanted to show that circular TALE proteins can be used in the fields of industry or medicine. After discussing multiple ways to simplify the use of TALEs, we came up with the idea of binding the TALE onto a membrane to allow it to be carried around easily and to be applied to medical tests like a blood test or analyzing other DNA-samples. As a proof of principle, we linked DNA onto the chip and a TALebot can later bind to this special DNA sequence.
First, a chip was spotted with oligonucleotides that contain the particular DNA sequence which our GFP-TALE can bind to. We used the spotter “GeSim Nanoplotter 2.1”. As a control, we spotted a chip that contains a different DNA sequence.
To generate a special pattern on the chip, we designed a program that was used to adjust the spotter in the desired way. This program can also be used to spot very different patterns and is described in detail in the Software part of our project.
The immobilized oligonucleotides will be hybridized with two kinds of samples. To test the system, we will hybridize it with Cy3 and Cy5 labeled oligonucleotides and scan the chips with a “GenePix Microarray Scanner 4000B”. Secondly, we planned to hybridize oligonucleotides that have been dimerized into double stranded DNA with our GFP-TALE and detected the GFP. The concept is that if the TALE binds to the DNA, we can later detect the GFP signal and see if the desired pattern is formed.