PART COLLECTION

Best part collection - a toolkit to functionalise BC with proteins

During the making of our project, we discovered that bacterial cellulose (BC) is an incredibly versatile material with a lot of applications. Because we are firm believers in collaborative science, we have developed a toolkit to further utilise this material. The goal of this part collection is to give future iGEM teams -or other researchers- a head start when developing new applications for BC.

This part collection consists of 11 (C3-13) composite parts, which are made up from twenty-two basic parts. Central to this part collection are the basic parts dCBD and cipA, the two cellulose binding domains (CBDs) investigated during this project. These parts are obtained from previous iGEM teams and adapted/applied in our toolkit (BBa_K1321340 and BBa_K4380000). dCBD is a family one CBD, which utilises disulphide bonds and aromatic residues for binding, and generally only binds to crystalline cellulose1. cipA is a family three CBD, which binds to both crystalline and amorphous cellulose using a large beta-strand framework2.

Other basic parts (B4 - B12) in this collection are reporter proteins, such as GFP, or functional proteins that we have used to modify the BC properties, such as HFBI to increase hydrophobicity, or to add properties, such as Cry3Aa to confer toxicity to beetle species. The basic parts B13-B22 are parts we used to correctly express our proteins, such as linker sequences and genes that make up our expression plasmids.

The first two composite parts in this collection are the two expression plasmids we used in our project, one for Escherichia coli and one for Saccharomyces cerevisiae. We decided to work with two different organisms to facilitate both ex situ and in situ protein functionalisation approaches. Proteins produced by E. coli can be purified using their His-tag and combined with BC after harvesting. Proteins produced by S. cerevisiae are secreted into the culture medium through their signal peptide and are incorporated into the BC while it grows. The remaining eleven composite parts are coding sequences ready to be inserted into these plasmids. This allows for quick and easy expression of these proteins. We highly encourage any future iGEM teams to expand upon this toolbox by adding more protein sequences to the toolbox that allow for different BC properties.

Composite parts

Nr. Name Nickname Used in Description Relevance
C1 BBa_259CTPBX pBCD5_KanR E. coli Plasmid backbone containing a T7 expression system, bicistronic design element, a kanamycin resistance gene, and a p15A origin of replication. This plasmid backbone can be used to express proteins in E. coli strains that have a T7 polymerase on their genome, such as the NiCo21 (DE3) strain that was used in this project. Additionally, the empty vector may also be used to validate transformation protocols.
C2 BBa_250N9BLI plasmid_CEN-ARS_KanMX S. cerevisiae Plasmid backbone no coding sequence for a functional protein. Contains the CEN/ARS ori and KanMX marker. This plasmid backbone can be used to express proteins in S. cerevisiae. Additionally, the empty vector may also be used to validate transformation protocols and act as a control.
C3 BBa_25OFBAY0 cipA_sfGFP E. coli Coding sequence for the expression of the cipA-sfGFP fusion protein in E. coli. Contains a His-tag for purification purposes. The cipA-sfGFP fusion protein was used to validate the binding affinity of the cipA domain to BC by measuring the strength of the fluorescent signal after treating BC with protein solution followed by a washing step.
C4 BBa_25SRDDT7 mUkG1_cipA_AGA2 S. cerevisiae Coding sequence for the expression of the mUkG1-cipA fusion protein in S. cerevisiae. Contains a AGA2 signal peptide for secretion. The mUkG1-cipA fusion protein was used to validate the binding affinity of the cipA domain to BC by measuring the strength of the fluorescent signal after treating BC with protein solution followed by a washing step.
C5 BBa_25258K5R mUkG1_dCBD_AGA2 S. cerevisiae Coding sequence for the expression of the mUkG1-dCBD fusion protein in S. cerevisiae. Contains a AGA2 signal peptide for secretion. The mUkG1-dCBD fusion protein was used to validate the binding affinity of the cipA domain to BC by measuring the strength of the fluorescent signal after treating BC with protein solution followed by a washing step.
C6 BBa_25MVPDX0 Spycatcher003_sfGFP_6xHis E. coli Coding sequence for the expression of the Spycatcher003-sfGFP fusion protein in E. coli. The spycatcher003 domain may be used to link the sfGFP to proteins containing the Spytag003 peptide. Contains a His-tag for purification purposes. The Spycatcher003-sfGFP fusion protein was used to validate and quantify the presence of the other fusion proteins produced by S. cerevisiae. Because these proteins all had a Spytag003 domain, this part could be covalently attached to them, which allowed for quantification of the sfGFP fluorescent signal.
C7 BBa_25N5WL5K cipA_Cry3Aa E. coli Coding sequence for the expression of the cipA-Cry3Aa fusion protein in E. coli. Contains a His-tag for purification purposes. The cipA-Cry3Aa fusion protein was used to make BC toxic to Coleoptera. The protein was immobilised on the seed coating which caused starvation effects, indicative of reduced feeding behaviour, in mealworms (Alphitobius diaperinus). This marks the first step towards using our seed coatings as a more sustainable alternative to classical pesticides.
C8 BBa_258PB1SY HFBI_cipA_AGA2 S. cerevisiae Coding sequence for the expression of the HFBI-cipA fusion protein in S. cerevisiae. Contains a AGA2 signal peptide for secretion. The HFBI-cipA fusion protein can be used to increase the hydrophobicity of BC. This can be advantageous for the delayed germination of seeds.
C9 BBa_25M93E07 HFBI_dCBD_AGA2 S. cerevisiae Coding sequence for the expression of the HFBI-dCBD fusion protein in S. cerevisiae. Contains a AGA2 signal peptide for secretion. The HFBI-dCBD fusion protein can be used to increase the hydrophobicity of BC. This can be advantageous for the delayed germination of seeds.
C10 BBa_25JO5U5D CBH1_cipA_AGA2 S. cerevisiae Coding sequence for the expression of the CBH1-cipA fusion protein in S. cerevisiae. Contains a AGA2 signal peptide for secretion. The CBH1-cipA fusion protein has a cellulase-like effect on BC. This means that the biodegradability of the material will be improved, which can speed up the release of active compounds contained in the coating.
C11 BBa_25FPHHHE CBH1_dCBD_AGA2 S. cerevisiae Coding sequence for the expression of the CBH1-dCBD fusion protein in S. cerevisiae. Contains a AGA2 signal peptide for secretion. The CBH1-dCBD fusion protein has a cellulase-like effect on BC. This means that the biodegradability of the material will be improved, which can speed up the release of active compounds contained in the coating.
C12 BBa_25WDECJ3 AmilCPblue_dCBD_AGA2 S. cerevisiae Coding sequence for the expression of the AmilCPblue-dCBD fusion protein in S. cerevisiae. Contains a AGA2 signal peptide for secretion. The AmilCPblue-dCBD fusion protein can stain the BC matrix with a blue colour, allowing us to change the colour of our seed coatings. From our human practices, we learned that colour is a very important factor for farmers, because a uniform colour signifies a high quality product.
C13 BBa_25FS93QF AmilGFPyellow_dCBD_AGA2 S. cerevisiae Coding sequence for the expression of the AmilGFPyellow-dCBD fusion protein in S. cerevisiae. Contains a AGA2 signal peptide for secretion. The AmilGFPyellow-dCBD fusion protein can stain the BC matrix with a yellow colour, allowing us to change the colour of our seed coatings. From our human practices, we learned that colour is a very important factor for farmers, because a uniform colour signifies a high quality product.

Basic parts

Nr. Name Nickname Used in Description Relevance
B1 BBa_K1321340 dCBD E. coli Synthetic double cellulose binding domain. Contains two domains from Trichoderma reesei cellobiohydrolases, spaced by a linker sequence. dCBD is a CBD belonging to family 1 of the CBDs. In this project, it was used to fuse several active proteins that were produced in S. cerevisiae to the surface of BC to change the material properties.
B2 BBa_25B74R7S cipA E. coli Cellulose binding domain from Clostridium thermocellum. cipA is a CBD belonging to family 3 of the CBDs. In this project it was used to fuse several active proteins that were produced in S. cerevisiae or E. coli to the surface of BC to change the material properties. This version of cipA is codon optimised for expression in E. coli.
B3 BBa_257HMYMW cipA S. cerevisiae Cellulose binding domain from Clostridium thermocellum. cipA is a CBD belonging to family 3 of the CBDs. In this project it was used to fuse several active proteins that were produced in S. cerevisiae or E. coli to the surface of BC to change the material properties. This version of cipA is codon optimised for expression in S. cerevisiae.
B4 BBa_25G620IT sfGFP E. coli Superfolder green fluorescent protein. Superfolder GFP is a variant of the GFP molecule that performs much better than its wild type variant when fused to a different protein domain. Whereas the wild type might have trouble correctly folding when fused to poorly folded polypeptides, sfGFP does not share these struggles, making it a popular choice in fusion protein production. In this project it was fused to cipA and Spycatcher003 to validate and quantify CBD binding affinity.
B5 BBa_25VKT7TL mUkG1 S. cerevisiae Monomeric variant of Umikinoko-Green fluorescent protein. mUkG1 is a green fluorescent protein derived from a soft coral. This fluorophore has been reported to perform much better in yeast than traditional GFP variants. For that reason, this protein was chosen to act as a reporter in our CBD yeast toolkit.
B6 BBa_250L082D Spytag003 E. coli Can undergo protein-protein interactions with its Spycatcher003 counterpart to form a covalent bond. This part was fused to cellulose-binding fusion proteins produced by S. cerevisiae. This allowed for detection and quantification of these proteins using Spytag003’s counterpart, Spycatcher003, fused to an sfGFP molecule.
B7 BBa_25A7C85Z Spycatcher003 E. coli Can undergo protein-protein interactions with its Spytag300 counterpart to form a covalent bond. This part was fused to an sfGFP molecule. This allowed for detection and quantification of cellulose-binding fusion proteins produced by S. cerevisiae, as these were fused to Spycatcher003’s counterpart, Spytag003.
B8 BBa_2544O00E Cry3Aa E. coli Insecticidal protein from Bacillus thuringiensis. The bt protein Cry3Aa is naturally produced by Bacillus thuringiensis subsp. tenebrionis and is toxic to members of the order Coleoptera. In this project, it was investigated to be used as a biological control to protect seeds from pest insects.
B9 BBa_254BBZE3 AmilCPblue S. cerevisiae Blue chromoprotein. AmilCPblue was fused to a cellulose binding domain to change the colour of the BC matrix. Although this is purely an aesthetic change, we have found this to be a very important factor for our customers.
B10 BBa_2538H5OR AmilGFPyellow S. cerevisiae Yellow chromoprotein. AmilGFPyellow was fused to a cellulose binding domain to change the colour of the BC matrix. Although this is purely an aesthetic change, we have found this to be a very important factor for our customers.
B11 BBa_25INM0JW HFBI S. cerevisiae Class II hydrophobin 1 (HFBI) is a small hydrophibic protein derived from Trichoderma reesei. HFBI was fused to a cellulose binding domain to make BC more hydrophobic, which can be useful for delayed germination purposes.
B12 BBa_25L5H7E5 CBH1 S. cerevisiae Exocellobiohydrolases 1 (CBH1) can catalyse the hydrolysis of 1,4-beta-D-glucosidic bonds in cellulose to release the disaccharide cellobiose, improving the biodegradability. CBH1 was fused to a cellulose binding domain to increase the biodegradability of BC. Increasing the biodegradability allows for faster release of embedded compounds.
B13 BBa_K1614000 T7 promoter E. coli Promoter from T7 bacteriophage. Very strong promoter, often used for protein expression. The T7 promoter was used to produce heterologous proteins in E. coli. Because it is controlled by a separate DNA-polymerase than the rest of the genome -which itself is placed under the control of an inducible promoter- it is possible to achieve very high protein titres.
B14 BBa_25KVSR5Q BCD5 E. coli Bicistronic design element. Facilitates binding of the ribosomes to the RNA, and thus protein production. Bicistronic design elements greatly improve protein production when compared to monocistronic design elements. It was used to achieve high protein titres.
B15 BBa_25X9RPLK Synthetic terminator E. coli Synthetic terminator consisting of a stem-loop and a T-stretch. This terminator was used to arrest transcription of the coding sequences on our production plasmid in E. coli.
B16 BBa_25U4ZG1K p15A_ORI E. coli Medium copy origin of replication. The p15A_ORI ensures replication of the plasmid alongside the genome. Plasmids with a p15A_ORI have a range of about 20-30 copies per cell.
B17 BBa_258UIRIC NeoR/KanR E. coli Gene that confers antibiotic resistance to neomycin and kanamycin. This gene makes bacteria immune to neomycin and kanamycin. This allows for selection of cells that have successfully integrated the plasmid, and ensures the plasmid is not lost over time.
B18 BBa_25ZYLAYB NEOKAN_promoter E. coli Constitutive promoter to continually express the NeoR/KanR gene. This strong constitutive promoter was used to ensure that the antibiotic resistance gene was always active.
B19 BBa_2563QDP3 (GGGGS)3 linker S. cerevisiae Flexible linker designed for fusion proteins. This linker was used to separate protein domains in a fusion protein, ensuring that their function is not affected by steric hindrance.
B20 BBa_25BVPI08 GSTG linker E. coli Short, rigid linker designed for fusion proteins. This linker is used to separate protein domains in a fusion protein, ensuring that their function is not affected by steric hindrance.
B21 BBa_K3033006 6xHIS E. coli Protein tag consisting of six histidine residues. The 6xHIS-tag was used for protein purification.
B22 BBa_25DJZF34 AGA2 S. cerevisiae The AGA2 signal peptide is used for protein secretion in yeast. AGA2 was fused to all fusion proteins produced in S. cerevisiae to allow for protein secretion into the medium.
(1)
Lehtiö, J.; Sugiyama, J.; Gustavsson, M.; Fransson, L.; Linder, M.; Teeri, T. T. The Binding Specificity and Affinity Determinants of Family 1 and Family 3 Cellulose Binding Modules. Proceedings of the National Academy of Sciences of the United States of America 2003, 100 (2), 484–489. https://doi.org/10.1073/pnas.212651999.
(2)
Tormo, J.; Lamed, R.; Chirino, A. J.; Morag, E.; Bayer, E. A.; Shoham, Y.; Steitz, T. A. Crystal Structure of a Bacterial Family-III Cellulose-Binding Domain: A General Mechanism for Attachment to Cellulose. The EMBO Journal 1996, 15 (21), 5739–5751.