Our results

Our project encompassed two main approaches: the cloning of coding sequences for our chosen biopolymers into plasmids for Pichia pastoris, and, simultaneously, making a variety of brick mixtures of different sizes and components, and testing them. Throughout our work, we adjusted our experiments based on troubleshooting according the engineering cycle and after gaining insights during our human practices analyses.

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The Cloning

We had varying success with out cloning, and will show our progress in the following order: spider silk, PHB and gelatin.

Main Strain Spider Silk

Insert

Since the insert can not be synthesized due to a high number of repeating sequences, it was built together by PCR. After a PCR-product in the desired size was retrieved, it was sent to the company “Microsynth” for sequencing.

Three further PCR-products, were created out of the first PCR-product, together creating the complete main strain spider silk sequence. The sequences of these PCR-products were also confirmed by sending them for sequencing to the company “Microsynth”.

Backbone 1

After receiving the correct sequences for the three PCR-products, the DNA-parts were cloned into an empty backbone 1 by Golden Gate. A first indication for a successful cloning is given by the comparison of the incubated E.coli plates of the negative control with the sample. Since no colonies could be observed on the negative control a successful cloning appeared to be likely.

A further sign for the correct plasmid was given by the control digest, showing bands at the correct sizes for most of the Mini-Prep samples. These samples were again sent to the company “Microsynth” for sequencing.

Backbone 2

Four backbone 2 versions were created by Golden Gate cloning of the backbone 1 plasmid, proven to obtain the correct sequence. Since the sequence of the insert was already confirmed by sequencing the backbone 1, only the comparison of the E.coli plates and the restriction digest of the Mini-Preps were needed as a evidence for a successful cloning.

Backbone 3

Three different versions of the backbone 3 were obtained, two containing one expression cassette each (Fig. 1 & 2), only differing from each other in the used promoters to create a direct comparison of the efficiency of these promoters. The third created backbone 3 version entails four different expression cassettes (Fig. 3). Similar to the backbone 2 versions, the successful cloning was proven by the comparison of the E.coli plates and the restriction digest of the Mini-Preps.

Protein Expression in P.Pastoris

The proteins encoded by the backbone 3 plasmids are expected to be secreted into the surrounding media, due to the secretion factor added to the backbone 1 plasmid. Even though the secreted protein has a molecular weight of approximately 28kDa, the main strain spider silk is suggested to build homodimers formed by two main strain spider silk monomers and therefore resulting into band sizes of approximately 56kDa [1].

In a first trial protein samples, gained from the Pichia pastoris clones using the pGAP and the pTEF promoter after 12, 24 and 36 hours were loaded on an SDS-PAGE gel (Fig.4). A distinctive band can be seen in all samples but not the wild type control at a size of approximately 56kDa which would be appropriate for the homodimers suggested to be built by the main strain spider silk protein.

Further SDS-PAGE gels will be shown at the Jamboree.

PHB

Backbone 1

To gain PHB, four different proteins need to be present in the final plasmid.

In a first step the synthesized inserts, each coding for a one of the four proteins needed for the PHB synthesis, were cloned into a backbone 1 by Golden Gate.

To ensure the successful cloning, the samples were sent to the company “Microsynth” for sequencing after first indications for a successful cloning were obtained by comparing the E.coli plates to the negative control and restriction digest.

Backbone 2

After the sequences were verified, the backbone 1 versions were cloned into backbone 2, adding various promoters and terminators to create four separate expression cassettes.

Since the sequences of the inserts were already confirmed in the backbone 1 versions, only a comparison of the E.coli plates to the negative control and a restriction digest were needed to prove the cloning success.

Backbone 3

All four backbone 2 versions were combined into one backbone 3 plasmid, to ensure all four proteins are produced by Pichia pastoris (Fig.5).

The successful cloning was proven by the comparison of the E.coli plates to the negative control and a restriction digest of the Mini-Preps, similar to the confirmation of the cloning success of the backbone 2 versions.

Gelatin

Already early on in the project, gelatin presented a challenge.

Over the passing weeks, we performed Golden Gate Assembly to clone our BB1 plasmid with the sequence that codes for gelatine and the alpha mating factor as an insert. After that, the plasmids were transformed into Ecoli which were then plated on LB-Kana agar plates. The plasmids were then later extracted.

To confirm that we did indeed have a correct plasmid we performed a digest with BglI and ran it on a 1% agarose gel. In one of our two samples, the bands were wrong, but the other one looked good, so we sent it to sequencing. The sequencing results confirmed that we have a correct plasmid.

We then took our BB1 plasmid and performed a Golden Gate Assembly into backbone 3, together with the promoter and the terminator. The assembled plasmid was transformed into Ecoli and plated on LB-Nat agar plates. After isolating the plasmids we performed a digest with PstI, but we don’t have the results for that yet.

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Our Bricks

We divided the mixtures of our bricks into three components: solid, binder and liquid.

For our first brick experiment we used river sand as a solid and also mixed it with lignin. As a binder we used gelatin and Agar-Agar, and the liquid was a) only BG11 medium b) BG11 medium with CaCO3 and 3) our Synechocystis culture. The function of 3) was not to let the Synechocystis precipitate CaCO3 but solely to see how the cell material is interacting with the mixture. We made five cubes of each combination and two sets of the experiment: we let them dry at RT and 4°C.

The full, detailed results of our first brick tests you can find here.