Parts

We chose to design our constructs with the pET28a vector since it was mentioned in the litterature for the expression of a recombinant green lacewing silk protein1. All the information about this common plasmid has been found on AddGene. We purchased it via GeneScript and visualized it in SnapGene.

pET28a is usually employed for bacterial gene expression with a high level. This plasmid composed of 5369 bp is constitutive, non-viral and transient. It contains:

• A selection marker: a kanamycin resistance gene to allow genetic selection of transformed E.coli. This procedure is carried out with a standard medium complemented with kanamycin antibiotic.

• An inducible expression system based on three components:

1. A T7 polymerase gene,chromosomally integrated in the E.coli BL21(DE3) cells genome, that can transcript the gene under the T7 promoter when expressed.
2. A T7 promoterpresent in the pET28a plasmid backbone and positioned before the target gene to induce its expression when the T7 polymerase gene is transcripted.
3. A lacI gene under lacI promoter that codes for a lac repressor, which is an inhibitor of the T7 polymerase gene’s expression. When IPTG is put, it enters in competition with the lac repressor and allows the expression of the T7 polymerase gene. It is present in the pET28a plasmid backbone.

• Additional features:

• 6xHis-tag.
• insert that can be replaced.

We did small changes in the standard backbone to optimize our constructs. We modified the 6xHis-tag by a 10xHis-tag and replaced the NcoI restriction site by a PmeI restriction site after the RBS sequence. To understand better why we did these modifications, check out our Engineering Choices section on the Design Page.

Our basic parts describe and document essential biobricks we used in our constructs:

• BBa_K4439000 is the DNA sequence of a single [AS] module which was isolated from the recombinant green lacewing silk protein N[AS]8C1.

• BBa_K4439001 is the DNA sequence of our designed recombinant silk protein, inspired by the one studied in Felix Bauer’s thesis1. It has only four [AS] modules instead of eight to allow an easier gene synthesis since repetitive elements are difficult to synthesize.

• BBa_K4439002 is the DNA sequence of the recombinant green lacewing silk protein N[AS]8C defined in Felix Bauer’s thesis1.

• BBa_K4439003 is the DNA sequence coding for avitag, a protein tag which can be biotinylated and thus allow the linking between our proteins.

• BBa_K4439004 is the DNA sequence of a monomer of streptavidin, used in our project as a solubilization tag as well as a key player in the biotinylation-linking between our proteins.

• BBa_K4439010 is the DNA sequence coding for GST, a standard solubilization tag.

• BBa_K4439011 is the DNA sequence coding for TEV cleavage site to get rid of the His-tag to enable the cross-linking of the AS modules.

• BBa_K4439012 is the DNA sequence of our plasmid construct 02b which codes for the SR-AS-TEV-10xHis fusion protein. This fire retardancy protein would be used to demonstrate the modularity of our protein coating.

• BBa_K4439015 is the DNA sequence of our plasmid construct 01b which codes for the SR-Avitag-10xHis fusion protein. This fire retardancy protein has been used to demonstrate the modularity of our protein coating.

Our composite parts gather all our plasmids’ constructs expressing our recombinant fusion proteins to tune the cellulose aerogel’s characteristics:

• BBa_K4439007 is the DNA sequence of our plasmid construct 01a which codes for the mSA-N[AS]4C-CBD-10xHis fusion protein. The latter is essential in the formation of a waterproof silk biofilm.

• BBa_K4439009 is the DNA sequence of our plasmid construct 02a which codes for the GST-N[AS]8C-CBD-10xHis fusion protein. The latter would be essential in the formation of a waterproof biofilm.

• BBa_K4439013 is the DNA sequence of our plasmid construct 03a which codes for the mSA-GFP-CBD-10xHis fusion protein. The latter has been used as control for the experiments performed on BBa_K4439007 and BBa_K4439008.

To understand better our five different plasmid constructs, check out the “Engineering Choices” section of our Design Page.