Parts
A part is a sequence of the DNA that encodes for a biological function. These parts can broadly be categorised into basic and composite parts. A DNA functional unit that cannot be subdivided into smaller components is referred to as a basic part. On the contrary, a composite part is a functional unit that can be subdivided, being made up of two or more basic parts. The table below shows the list of basic parts that were used throughout our project. Our project aslo employed a vector backbone, pET45b, for cloning and protein expression. The vector includes a T7 promoter (BBa_I719005), RBS, His-tag and kanamycin resistance gene; all of which were integral to the purification of our protein. We do not include on pET45b in our list of parts as it contains restriction sites that are not compatible with iGEM standards. Instead, we focus on the parts that make up our fusion protein constructs.
Name Type Description Designer Length (bp)
BBa_K4190006 Coding EK Cleavage site NA 15
BBa_K3033009 Coding eGFP Coding region NA 717
BBa_K1159001 Coding NLuc Coding region NA 510
BBa_J18918 Coding TEV Cleavage site NA 21
BBa_K4658000 Coding Linker peptide Farouk, Sonja 51
BBa_K4658001 Coding AviTag spacer Farouk, Sonja 15
BBa_K4658002 Coding AviTag biotinylation site Farouk, Sonja 45
BBa_K4658003 Coding Anchor peptide Farouk, Sonja 141
BBa_K4658004 Composite NLuc construct Farouk, Sonja 796
BBa_K4658005 Composite eGFP construct Farouk, Sonja 1000

The linker peptide is a 51 bp long peptide made up predominantly of alanine and lysine residues that is used to create a link between the anchor peptide and eGFP or Nluc. This peptide was used to connect the peptide with the signal protein to form the fusion protein.

Sequence and Features:

5'-GGCTTTCGCTGCTGCTTCTTTGGCGGCCGCTTCTTTCGCGGCCGCTTCCGC-3'

The Avitag spacer is a 15 bp short peptide used to provide binding space between the AviTag (BBa_K4658002) and the anchor peptide (BBa_K4658003) for biotinylation with the BirA enzyme.

Sequence and Features:

5'-CGCCGCCGCCGCCGC-3'

The AviTag, also known as an acceptor peptide, is a 45-bp peptide used for highly specific enzymatic biotinylation. We aimed to use the AviTag to biotinylate the anchor peptide to magnetic beads for our sandwich assay. Since, our anchor peptide consisted of multiple lysine residues using the chemical biotinylation had the potential to destroy the protein.

Sequence and Features:

5'-TTCATGCCATTCAATTTTCTGCGCTTCAAAAATATCGTTCAGGCC-3'

The anchor peptide (LCI) is a polymer binding peptide, which was used for the binding of the fusion protein to polypropylene. To confirm the specific binding of our anchor peptide to polypropylene, we used the fusion protein constructs and measured the fluorescence/luminescence after washing.

Sequence and Features:

5'-TTTGCGATCCCACACTTCATAAATGCCCACCCAATAGCCTTTGCTGCTATCATAATATTTGCTTTT AAAAATCCATTTGGTGCCATCCAGCACAAAGCTCGCCGCAAAGTTGCCGTTCGGGCTCTGCACCAG TTTAATCGC-3'

This NLuc fusion protein was created for the binding of microplastics, specifically polypropylene, using an anchor peptide and NLuc enzyme. This composite part includes the Anchor peptide, AviTag spacer, AviTag Biotinylation Site, Linker Peptide, TEV Cleavage site, and NLuc coding region (as described above).

Characterisation:

Restriction digestion was carried out using HindIII and SacI restriction enzymes for both the NLuc and eGFP constructs. Cloning success was confirmed with sequencing. Protein expression and purification was carried out using E. coli BL21 cells. Expression was induced with 0.5mM IPTG until the OD reached 0.5-0.6. This was followed by overnight induction at 25 and 37°C.

Sequence and Features:

5'-AGCTTTTATTTGCGATCCCACACTTCATAAATGCCCACCCAATAGCCTTTGCTGCTATCATAATA TTTGCTTTTAAAAATCCATTTGGTGCCATCCAGCACAAAGCTCGCCGCAAAGTTGCCGTTCGGGC TCTGCACCAGTTTAATCGCCGCCGCCGCCGCCGCTTCATGCCATTCAATTTTCTGCGCTTCAAAA ATATCGTTCAGGCCGCCCTGAAAATACAGGTTTTCGGCTTTCGCTGCTGCTTCTTTGGCGGCCGC TTCTTTCGCGGCCGCTTCCGCCGCCAGAATGCGTTCGCACAGCCGCCAGCCGGTCACTCCGTTGA TGGTTACTCGGAACAGCAGGGAGCCGTCGGGGTTGATCAGGCGCTCGTCGATAATTTTGTTGCCG TTCCACAGGGTCCCTGTTACAGTGATCTTTTTGCCGTCGAACACGGCGATGCCTTCATACGGCCG TCCGAAATAGTCGATCATGTTCGGCGTAACCCCGTCGATTACCAGTGTGCCATAGTGCAGGATCA CCTTAAAGTGATGATCATCCACAGGGTACACCACCTTAAAAATTTTTTCGATCTGGCCCATTTGG TCGCCGCTCAGACCTTCATACGGGATGATGACATGGATGTCGATCTTCAGCCCATTTTCACCGCT CAGGACAATCCTTTGGATCGGAGTTACGGACACCCCGAGATTCTGAAACAAACTGGACACACCTC CCTGTTCAAGGACTTGGTCCAGGTTGTAGCCGGCTGTCTGTCGCCAGTCCCCAACGAAATCTTCG
AGTGTGAAGACGAGCT-3'

This eGFP fusion protein was created as a control for the micrplastic binding assay using an anchor peptide and eGFP protein. This composite part includes the Anchor peptide, AviTag spacer, AviTag Biotinylation Site, Linker Peptide, TEV Cleavage site, and eGFP coding region (as described above).

Characterisation:

Restriction digestion was carried out using HindIII and SacI restriction enzymes for both the NLuc and eGFP constructs. Cloning success was confirmed with sequencing. Protein expression and purification was carried out using E. coli BL21 cells. Expression was induced with 0.5mM IPTG until the OD reached 0.5-0.6. This was followed by overnight induction at 25 and 37°C.

Sequence and Features:

5'-AGCTTTTATTTGCGATCCCACACTTCATAAATGCCCACCCAATAGCCTTTGCTGCTATCATAATA TTTGCTTTTAAAAATCCATTTGGTGCCATCCAGCACAAAGCTCGCCGCAAAGTTGCCGTTCGGGC TCTGCACCAGTTTAATCGCCGCCGCCGCCGCCGCTTCATGCCATTCAATTTTCTGCGCTTCAAAA ATATCGTTCAGGCCGCCCTGAAAATACAGGTTTTCGGCTTTCGCTGCTGCTTCTTTGGCGGCCGC TTCTTTCGCGGCCGCTTCCGCTTTATACAGTTCATCCATGCCCAGGGTAATGCCCGCCGCGGTCA CAAATTCCAGCAGCACCATATGATCGCGTTTTTCGTTCGGATCTTTGCTCAGCGCGCTCTGGGTG CTCAGATAATGGTTATCCGGCAGCAGCACCGGGCCATCGCCAATCGGGGTGTTCTGCTGATAATG ATCCGCCAGCTGCACGCTGCCATCTTCAATGTTATGGCGAATTTTAAAGTTCACTTTAATGCCGT TTTTCTGTTTATCCGCCATAATATACACGTTATGGCTGTTATAGTTATATTCCAGTTTATGGCCC AGAATGTTGCCATCTTCTTTAAAATCAATGCCTTTCAGTTCAATGCGGTTCACCAGGGTATCGCC TTCAAATTTCACTTCCGCGCGGGTTTTATAGTTGCCATCATCTTTAAAAAAAATGGTGCGTTCCT GCACATAGCCTTCCGGCATCGCGCTTTTAAAAAAATCATGCTGTTTCATATGATCCGGATAGCGG CTAAAGCACTGCACGCCATAGGTCAGGGTGGTCACCAGGGTCGGCCACGGCACCGGCAGTTTGCC GGTGGTGCAAATAAATTTCAGGGTCAGTTTGCCATAGGTCGCATCGCCTTCGCCTTCGCCGCTCA CGCTAAATTTATGGCCGTTCACATCGCCATCCAGTTCCACCAGAATCGGCACCACGCCGGTAAAC
AGTTCTTCGCCTTTGCTCACGAGCT-3'
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