Name	Type	Short Description	Description	Designers	Length	Description in the Registry
BBa_K5077000	Coding	Resilin exon optimized	The exon we used for the Repeatigo method.	Luna Brandes, Greta Stoltmann, Boy Hansen	45 bp	The exon we used for the Repeatigo method. The resilin sequence is a single exon that has been optimised for utilisation in the Repeatigo method, given its particular requirements.
BBa_K5077001	Coding	Resilin Oligo 1 Repeat	One of the six Oligonucleotides needed for the Repeatigo method	Luna Brandes, Greta Stoltmann, Boy Hansen	45 bp	The aim was to generate 64 repeats of the resilin sequence (BBa_K5077000). In order to achieve this, the Repeatigo method was developed. Six distinct Oligonucleotides are necessary for the Repeatigo method. This specific Oligonucleotide is one of them. Further details can be found in the description of part BBa_K5077019.
BBa_K5077002	Coding	Resilin Oligo 2 Repeat	One of the six Oligonucleotides needed for the Repeatigo method	Luna Brandes, Greta Stoltmann, Boy Hansen	45 bp	The aim was to generate 64 repeats of the resilin sequence (BBa_K5077000). In order to achieve this, the Repeatigo method was developed. Six distinct Oligonucleotides are necessary for the Repeatigo method. This specific Oligonucleotide is one of them. Further details can be found in the description of part BBa_K5077019.
BBa_K5077003	Coding	Resilin Oligo NdeI 1 end	One of the six Oligonucleotides needed for the Repeatigo method	Luna Brandes, Greta Stoltmann, Boy Hansen	21 bp	The aim was to generate 64 repeats of the resilin sequence (BBa_K5077000). In order to achieve this, the Repeatigo method was developed. Six distinct Oligonucleotides are necessary for the Repeatigo method. This specific Oligonucleotide is one of them. Further details can be found in the description of part BBa_K5077019.
BBa_K5077004	Coding	Resilin Oligo NdeI 2 end	One of the six Oligonucleotides needed for the Repeatigo method	Luna Brandes, Greta Stoltmann, Boy Hansen	43 bp	The aim was to generate 64 repeats of the resilin sequence (BBa_K5077000). In order to achieve this, the Repeatigo method was developed. Six distinct Oligonucleotides are necessary for the Repeatigo method. This specific Oligonucleotide is one of them. Further details can be found in the description of part BBa_K5077019.
BBa_K5077005	Coding	Resilin Oligo SacI 1 end	One of the six Oligonucleotides needed for the Repeatigo method	Luna Brandes, Greta Stoltmann, Boy Hansen	23 bp	The aim was to generate 64 repeats of the resilin sequence (BBa_K5077000). In order to achieve this, the Repeatigo method was developed. Six distinct Oligonucleotides are necessary for the Repeatigo method. This specific Oligonucleotide is one of them. Further details can be found in the description of part BBa_K5077019.
BBa_K5077006	Coding	Resilin Oligo SacI 2 end	One of the six Oligonucleotides needed for the Repeatigo method	Luna Brandes, Greta Stoltmann, Boy Hansen	46 bp	The aim was to generate 64 repeats of the resilin sequence (BBa_K5077000). In order to achieve this, the Repeatigo method was developed. Six distinct Oligonucleotides are necessary for the Repeatigo method. This specific Oligonucleotide is one of them. Further details can be found in the description of part BBa_K5077019.
BBa_K5077007	Plasmid_Backbone	pMK1	Plasmid with just one restriction site for each of the type II restriction enzymes BsaI, BsmBI and SacI	AG Deuerling; University of Constance	3409 bp	The plasmid was generated by a research group at the University of Constance. This plasmid was selected for the straightforward generation of multiple copies of the resilin gene (BBa_K5077000) via type II restriction enzymes. The plasmid has a single restriction site for each of the enzymes type II (BsaI, BsmBI, SacI).
BBa_K5077008	Coding	Resilin Repeat 1	One of the four Resilin repeats we used for the appraoch with the type II restriction enzymes	Hendrik Franke, Lisa Siemers, Boy Hansen	45 bp	This is one of the four Resilin repeats that were used for the Restriction Enzyme Type II method. These enzymes (BsaI, BsmBI, SacI) were used to create 64 repeats of resilin within one vector, to express a whole protein made of a long resilin strand.
BBa_K5077009	Coding	Resilin Repeat 2	One of the four Resilin repeats we used for the appraoch with the type II restriction enzymes	Hendrik Franke, Lisa Siemers, Boy Hansen	45 bp	This is one of the four Resilin repeats that were used for the Restriction Enzyme Type II method. These enzymes (BsaI, BsmBI, SacI) were used to create 64 repeats of resilin within one vector, to express a whole protein made of a long resilin strand.
BBa_K5077010	Coding	Resilin Repeat 3	One of the four Resilin repeats we used for the appraoch with the type II restriction enzymes	Hendrik Franke, Lisa Siemers, Boy Hansen	45 bp	This is one of the four Resilin repeats that were used for the Restriction Enzyme Type II method. These enzymes (BsaI, BsmBI, SacI) were used to create 64 repeats of resilin within one vector, to express a whole protein made of a long resilin strand.
BBa_K5077011	Coding	Resilin Repeat 4	One of the four Resilin repeats we used for the appraoch with the type II restriction enzymes	Hendrik Franke, Lisa Siemers, Boy Hansen	45 bp	This is one of the four Resilin repeats that were used for the Restriction Enzyme Type II method. These enzymes (BsaI, BsmBI, SacI) were used to create 64 repeats of resilin within one vector, to express a whole protein made of a long resilin strand.
BBa_K5077014	Coding	hasA	This is a hyaluronic acid synthase, which can be employed for the production of hyaluronic acid via E. coli.	Lena Abeln	1251 bp	This is a hyaluronic acid synthase, which can be employed for the production of hyaluronic acid via E. coli.
BBa_K5077015	Plasmid_ Backbone	pUC with araC and AmpR	This plasmid backbone can be used for multiple cloning approaches. It has a araC site, as well as a Ampicilin resistance.	Lena Abeln	3999 bp	The plasmid backbone is suitable for use in a number of different cloning approaches. The ampicillin resistance enables the differentiation between transformed and non-transformed cells, while the araC site facilitates the regulation of gene expression.
BBa_K5077017	Plasmid_Backbone	pET-28c(+)	A variant of the pET28a(+), which is a standard expression vector with a T7 promotor, two 6x His-tags with thrombin- and enterokinase restriction sites, and a kanamycin resistance.	Katharina Veith	5324 bp	The plasmid backbone of the pET-28c(+) vector is illustrated here. The pET-28c(+) vector is similar to the pET28a(+) vector which is a frequently used expression vector in E. coli. The plasmid backbone contains a T7 promoter that can be controlled with precision via IPTG. To facilitate the purification of the protein, the vector incorporates a His-tag and thrombin- and enterokinase restriction sites, which can be employed to remove the His-tag following the purification process. The kanamycin resistance enables the differentiation between transformed and non-transformed cells.
BBa_K5077018	Coding	EGFP + Linker + Overhangs	The EGFP gene was linked with a linker and overhangs to put that into the pUC-HasA vector via homologous recombination.	Boy Hansen, Malcolm Summer Rose-Heine	762 bp	This part was developed for the purpose of homologous recombination of pUC-HasA (BBa_K5077015, BBa_K5077014) and EGFP (BBa_J428076). The objective was to observe the production of hyaluronic acid synthase within the cells. Consequently, we attempted a process of homologous recombination, which necessitated the inclusion of the EGFP gene with overhangs. The linker was incorporated to facilitate the correct folding of HasA.

Name	Type	Short Description	Description	Designers	Length	Description in the Registry
BBa_K5077012	Coding	RE4	All four Resilin repeats (Resilin repeat 1-4) together	Hendrik Franke, Lisa Siemers, Boy Hansen	180 bp	This composite part illustrates the four resilin repeats that were employed in the restriction enzyme type II method. The objective was to generate 64 repeats of resilin, and these four repeats were to serve as the basis for numerous restriction ligation reactions with BsaI, BsmBI, and SacI enzymes to reach the desired amount.
BBa_K5077013	Plasmid	pMK1_RE4	The pMK1 plasmid with four Resilin repeats inside. This was part of the approach via type II restriction enzymes	Hendrik Franke, Lisa Siemers, Boy Hansen	3589 bp	This whole plasmid was used to generate a total of 64 repeats of the Resilin repeats. Four repeats are already there and the aim was to reach the 64 repeats via restriction ligation with restriction enzymes type II (BsaI, BsmBI, SacI).
BBa_K5077016	Plasmid	pUC_hasA_EGFP	The plasmid may be employed for the expression of a hyaluronic acid synthase fusion protein with enhanced green fluorescent protein (EGFP) in E. coli cells.	Boy Hansen, Malcolm Summer Rose-Heine, Lena Abeln	5981 bp	The plasmid may be employed for the expression of a hyaluronic acid synthase fusion protein with enhanced green fluorescent protein (EGFP) in E. coli cells.
BBa_K5077019	Plasmid	pET28c(+) with 64 resilin repeats	The pET28c(+) plasmid with 64 repeats of the optimized Resilin exon as an insert. The insert was created using the Repeatigo method.	Greta Stoltmann, Boy Hansen	8204 bp	siehe unten

We would like to submit a nomination for our plasmid pET28c(+) with 64 resilin repeats (BBa_K5077019)(see Fig. 1). The plasmid can be used to express 64 repeats of resilin exons, resulting in the formation of large resilin threads. These threads can be used to create a hydrogel via crosslinking with hyaluronic acid. The plasmid was originally derived from Katharina Veith, while the insert was generated using the Repeatigo method.

This plasmid represents a pivotal component of our project. The concept of using a hydrogel as a wound dressing for burn wounds is contingent upon the ability to create Resilin threads of sufficient length to crosslink them with chemically modified hyaluronic acid.

The plasmid itself represents a significant accomplishment. To achieve this level of repetition, a variety of techniques were employed, as a straightforward PCR approach was insufficient. The most promising approach was the Repeatigo method. To create this method, six different oligos were designed and allowed to anneal. Following this, a ligase was used to bind all of the oligos together. The oligos were designed to introduce NdeI and SacI restriction sites at the end and at the front of the whole sequence. The insert was then cloned with these two enzymes into the pET28c(+) vector and transformed into our E. coli cells. (read more about Repeatigo here)

For a more detailed examination of the results and accomplishments associated with this vector, we invite you to visit our results page. Should you require further technical data regarding the plasmid and its creation, we would invite you to consult the relevant page, accessible via the following link: Results