To verify our circularized TALE, we added the Strep Tag by Golden Gate Cloning. We used the Strep Tag for both the purification of the vector and for detection. We decided on a Strep-Tag because this Tag does not have to be on the end of a protein so it works even if the protein is circularized. If we would add other Tag sequences e.g. an His-Tag, some can only work if they are on the end of a protein.
In the one vector (iGem_02_Ax7R-RR) we used the Strep-Tag II which binds Strep-Tactin, an engineered form of streptavidin. Here are two sequences of this Tag, .they are all 24 bp.
In the other vectors (iGEM_02_Ax7L-DS, iGEM_02_GFP Hax3-2xNG and iGEM_02 GFP Hax3-2xNN) we used the Strep-Tactin(R)XT which enables new applications in the field of high throughput screening, batch purification, purification using denaturing conditions and protein interaction studies. It is 90 bp.
Inteins were integrated into proteins. They are used for the circularization of a protein by ligating the N- and C-terminal ends. The Heidelberg 2014 team already used them for their circulation but we changed the sequence to make the circularization more accurate. They successfully showed that circularization enhances resistance against proteases and denaturation. Furthermore, we show that circularization with a TAL-effector makes them more solid and durable for the everyday laboratory. If you want more Information to have a look at our results below. The Intein sequence has a size of 309 bp.
The eGFP is a part of our vector which synthesizes a green fluorescence protein (GFP). The origin of the GFP is the jellyfish Aequorea victoria. When exposed to blue or UV light, it emits a bright green light. The enhanced GFP (eGFP) has a higher intensity emission than the wild type GFP. Today the GFP is an essential fluorescence reporter protein for biology especially cell biology. It can be connected to other proteins to detect the expression of these proteins and their location.
We used the eGFP to detect our TAL effectors on a chip, spotted with oligonucleotides. these oligonucleotides include the target sequence of the TALE Hax3 – 2xNN (A C A C C C G/A G/A N C A)
The TEV site is a DNA Sequence encoding an amino acid sequence, which is cut by the TEV protease. The TEV protease is a protease with a high sequence specificity. The origin of the TEV protease is the tobacco each virus.
We use the TEV Site to get our circularized TALE protein back into a linear form. Only the linear TAL effector protein can bind the target sequence.
This part synthesizes a transcription activator-like (TAL) effector protein. They were found in Xanthomonas bacteria. These bacteria are plant pathogens and infect plants via a secretion type III system. In the plant cells, these proteins bind promoter sites. This leads to a transcriptional activation of the genes behind the promoter.
The proteins contain tandem repeats up to 34.5. Each repeat binds one nucleotide of the target sequence. The 12th and 13th amino acid of each repeat lead to the specific binding of the DNA sequence.
We circularized the TAL-effectors with the help of Inteins at the N- and C- Terminus of each TAL-effector.
The 12th and 13th amino acid of each of the repeats from our TALE Ax7L-DS are: NI NN HD NI HD NG NI NG NI NG NI NI NI HD HD HD HD HD
They bind the DNA sequence: A G/A C A C T A T A T A A A C C C C C
The 12th and 13th amino acid of each of the repeats from our TALE Ax7R-RR are: NG NN NI NI NN NN HD NG NG NN NI NG NN NI NN HD NG
They bind the DNA sequence: T G/A A A G/A G/A C T T G/A A T G/A A G/A C T
The 12th and 13th amino acid of each of the 11.5 repeats from our TALE Hax3 – 2xNG (B) are: NI HD NI HD HD HD NG NG NS HD NI NG.
They bind the DNA sequence: A C A C C C T T N C A T
The 12th and 13th amino acid of each of the 11.5 repeats from our TALE Hax3 – 2xNN are: NI HD NI HD HD HD NN NN NS HD NI
They bind the DNA sequence: A C A C C C G/A G/A N C A.