iGEM Rotterdam HR 2019

Biobrick Parts

Basic Parts

Four parts have been developed. These parts form the fundament of our project and will be briefly described below. All new biobrick parts have been flanked with RFC10 prefix and suffix.

GIGA-BLOCK 1 [BBa_K2993000]

This biobrick is called GIGA-BLOCK because it encodes for the fusion of a large protein array to a split enzyme. This biobrick consists of three parts:

ZincFinger-array 1
GSAT-linker
C-TEV split enzyme

The zinc finger-array is a multi-protein complex designed with the software of the company called Scripps Research (SR). SR has a website called: https://www.scripps.edu/barbas/zfdesign/zfdesignhome.php. This website helped us design specific zinc finger-arrays and their respective DNA-target. The zinc finger-array consists of six zinc fingers. Each zinc finger will bind a specific DNA-target.

The GSAT-linker is a peptide linker used to connect two peptides. The design of this linker was taken from biobricks part BBa_k404301 which was submitted by FreiGEM 2010. This part has an RFC standard RFC25. This is important to maintain the reading frame. This part was chosen because it encodes amino acids which do not interact with amino acids in their environment. The amino acids glycine and serine are zwitterionic and hydrophile - these properties make them a good choice for the repetitive sequence of the linker.

The C-TEV enzyme is, as the name suggests, the second half of the complete TEV- protease. The C-TEV is also originally taken as an already existing biobrick. It comes from the TU-Munich 2013 team. The biobrick number is BBa_K1159102. The team proved that this part works when reunited with its other half: the N-TEV enzyme. This split TEV will bind to its other half and form a functional TEV protease, which is needed to cleave off an inhibitor from β-lactamase.

GIGA-BLOCK 2 [BBa_K2993003]

This biobrick is called GIGA-BLOCK because it encodes for the fusion of a large protein array to a split enzyme. This biobrick consists of three parts:

ZincFinger-array 2
GSAT-linker
N-TEV split enzyme

The zinc finger-array is a multi-protein complex designed with the software of the company called Scripps Research (SR). SR has a website called: https://www.scripps.edu/barbas/zfdesign/zfdesignhome.php. This website helped us design specific zinc finger-arras and their respective DNA-target. De zinc finger-array consists of six zinc fingers. Each zinc finger will bind a specific DNA-target.

The GSAT-linker is a peptide linker used to connect two peptides. The design of this linker was taken from biobricks part BBa_k404301 which was submitted by FreiGEM 2010. This part has an RFC standard RFC25. This is important to maintain the reading frame. This part was chosen because it encodes amino acids which do not interact with amino acids in their environment. The amino acids glycine and serine are zwitterionic and hydrophile - these properties make them a good choice for the repetitive sequence of the linker.

The N-TEV enzyme is, as the name suggests, the first half of the complete TEV- protease. The N-TEV is also originally taken as an already existing biobrick. It comes from the TU-Munich 2013 team. The biobrick number is BBa_K1159101. The team proved that this part works when reunited with its other half: the C-TEV enzyme. This split TEV will bind to its other half and form a functional TEV protease, which is needed to cleave off an inhibitor from β-lactamase.

DNA-bridge [BBa_K2993004/BBa_K2993005/BBa_K2993006]

This part is unique to the zinc fingers that have been designed. With the zinc finger tools website, we have developed a unique zinc finger array with its respective DNA-target. This target sequence is attached to a DNA-bridge. This DNA-bridge contains 4 restriction sites for two specific enzymes. The idea is that we can test the optimal binding distance needed for the split-TEV protease to form a functional protease. By cutting with either one of the two, or both, there are three distances which can be tested for binding of the protease. This method can also be used to research the binding efficiency of the zinc finger to their target. If it does not bind well, a different array can then be made and tested out on a different DNA-bridge with the same method used as before. If it does work, the aptamers for a certain target can be ordered.

TEV-cleaving site-β-lactamase

This part plays a big role when it comes to the visual reaction change that the consumers want to see. As described in our main page, the β-lactamase will interact with nitrocefin, a chemical compound. This compound has a natural yellow color. In the presence of β-lactamase, an interaction between the two will occur, which will bring forth a visual color change from yellow to red. This will only happen when the inhibitor has been cleaved off. That can only be done by fusing the inhibitor (an unspecific protein sequence) with a TEV-cleaving site. This site will function as the recognition sequence for active TEV protease. And TEV will only be active when the aptamers have come in contact with the target of interest.

Pink and purple chromoproteins

For the bronze criteria, we had to perform characterization tests to an already existing biobrick part. We have chosen to work with two chromoproteins.

The chromoproteins that were used have the biobrick numbers:

BBa_K1033917 (Purple)
BBa_K1033926 (Pink)

The characterization tests that were performed, were tests regarding the protein stability under different pH-conditions.