With our project we introduce several new basic and composite parts. A list of all of them can be found on our parts page. Here we would like to emphasize two expression systems that can easily be used by other teams in the future.
Both of them pose the possibility to produce the two main components for a sustainable hydrogel made out of natural materials.

Resilin expression vector

The first composite part is the plasmid pET28c(+) containing 64 consecutive repeats of the resilin exon 1 (BBa_K5077019). With this plasmid we provide an expression system for the production of resilin, which will be ready-to-use for obtaining a hydrogel via crosslinking.
Future teams are invited to replicate this expression system in their own lab using our Repeatigo system which is further elucidated down below.

HasA expression system for hyaluronic acid production

The second component for building the sustainable hydrogel is hyaluronic acid (HA). Unlike resilin, HA is no protein and its production in Escherichia coli is reliant on the expression of the HasA synthase which complements the metabolism pathway in E. coli to produce HA as a final metabolic product. For incorporation of HasA into E. coli, we designed our pUC_hasA_EGFP plasmid (BBa_K5077016). As its name predicts, we incorporated the coding sequence for enhanced green fluorescent protein (EGFP) into it by genetically fusing the EGFP to the HasA synthase. Thus, we provide a reporter system to assess whether HasA is expressed in culture and whether HA could be produced. It can be used initially to optimize the added arabinose concentration for HasA expression. A green fluorescent culture - easily to be tested by using a plate reader - means HasA synthase is expressed and the culture can be used to purify HA. No fluorescence means the expression did not work and it is not worth it to purify the culture for HA.
These two expression vectors can be used by future iGEM teams to express resilin and to produce HA.

Reapeatigo - Cloning Repeats with Oligos - is a method we developed for cloning consecutive repeats that can not be synthesized. Since we struggled a lot cloning resilin and were running out of time using classic restriction-ligation cloning methods, we were in need for a fast and easy method to get our 64 resilin repeats.
After some Human Practices and some experimental design we developed the Repeatigo method. In short this method builds upon the assembly of 6 different types of oligos: two of them will form the main body of the repeats and the other 4 will assemble at the ends at both sides. These end oligos incorporate two different restriction recognition sites to enable controlled ligation into a vector.
Repeatigo gives future iGEM teams the chance to work with protein polymers that were very difficult to clone until now. There are many projects that could be based on the use of our new method. We are excited to see further developments!
Read more about Repeatigo on our Project Description page as well as on our part page for the resilin vector (BBa_K5077019) that we cloned using this method.

Throughout our project we had several opportunities to reach out to the public and developed new educational concepts. We now would like to hand over these concepts to future iGEM teams.
These concepts include:

• A muppet theater script: this script can be used to educate children about the importance of hygiene. We provided a German and an English version. Feel free to translate it in your native language!
• Coloring pictures of bacteria: We've created some amazing pictures of bacteria that the children can color in! This is a fantastic way to introduce younger children to the shapes of bacteria.
• Enzyme Puzzle: We've created some lovely templates for the four most important salivary enzymes: amylase, lipase, lysozyme and peroxidase. This helps children to understand the relationship between enzyme and substrate. All future iGEM teams have to do is to print out the template, cut it out and then glue it onto cardboard and cut it once again. And just like that the enzyme puzzle is ready to use!
• The crazy hydrogel game: an adapted version of the game “The Crazy Labyrinth”. Our instructions can be used and changed by future iGEM teams to explain different components of their own projects.
• Protocols and presentations for organizing science days: we provide protocols and presentations from the science days we organized for students. These experiments include the extraction of DNA of tomatoes and a demonstration of agroinfiltration. The presentations can be used for safety instructions and introductions to Genetic Engineering and Molecular Biology.

We are looking forward to see some variations of our concepts in the future!