iGEM Leiden | 2019

S.P.L.A.S.H.

Suckerin Polymer Layer to Achieve Sustainable Health

Demonstrate

Annually millions of people suffer debilitating burn wounds. As described in our human practices, there is a big gap in the current treatment methods of these wounds. Therefore, iGEM Leiden focussed on filling this gap by producing a hydrogel made of the suckerin protein. To achieve this, several experiments were designed and conducted.

We have produced significant amounts of suckerin-8, -9, -12, and -19 with different molecular properties and characteristics, which can be used to create a wide variety of hydrogels [1]. Creating these suckerins is already a big step into the direction of creating the optimal suckerin-based hydrogel. The use of specific suckerins, as the basis of a hydrogel, can accommodate each patient’s needs. Besides this, we have developed a production plasmid for improved protein production in Escherichia coli (E. coli). These accomplishments are the first essential step in the bulk production of the “smart” suckerin-based hydrogel, and further opportunities for infection prevention. Here, we demonstrate our production of suckerin.

Suckerin production in E. coli

The first step of the production of the bioactive (“smart”) hydrogel, was to produce the suckerin protein in a production host. There are various members of the suckerin protein family. Each one has a specific ratio of the two main regions of amino acids: the M1 and M2 modules. This varying composition of these modules results in distinct differences in length and properties between the final proteins. One of the suckerin proteins (suckerin-19) was already produced in E. coli under the inducible regulation of the Lac promoter [2]. During our research, we have proven that it is possible to additionally produce other suckerin proteins in E. coli. We chose to focus our project on the production of suckerin-8 (BBa_K3041015), suckerin-9 (BBa_K3041016), and suckerin-12 (BBa_K3041017) under the control of the Lac expression cassette (BBa_K314103). Suckerin-12 has a similar genetic composition as suckerin-19 but has a smaller molecular weight. Suckerin-8 and -9 mainly contain M1 or M2 modules, respectively, and belong to other evolutionary clades. Therefore, we decided that a comparison between these four suckerin proteins would be interesting in our research.

The genes were checked to ensure they are in the correct reading frame, amplified with PCR (Fig. 1), and inserted into the pBS1A3 plasmid. Transformants were checked by PCR, after transformation into the E. coli strain Rosetta.

Figure 1. Amplified suckerin genes 8, 9, and 12.
Polymerase chain reaction (PCR) products amplified with general prefix and suffix primers, shown by gel electrophoresis. A 1kb ladder (lane 1) was used. PCR product at different concentrations of suckerin-8 (408 bp, lane 2 and 3), suckerin-9 (568 bp, lane 4 and 5), and suckerin-12 (696 bp, lane 6 and 7). The gel confirms the amplification of the desired suckerin proteins.

Next, the bacteria were grown in one-liter bioreactors and the proteins were subsequently purified via inclusion body purification. After purification, the samples were visualized on SDS-PAGE gels (Fig. 2), with visible bands at the size of the corresponding suckerins [1]. This indicates that successful production of the different suckerin proteins was achieved.

Figure 2. On SDS-PAGE gel there is visible suckerin protein purified from E. coli, for each of the different suckerin proteins.
Ladder (lane 1), suckerin-12 (23 kDa, lane 3) , suckerin-9 (19 kDa, lane 4) and suckerin-8 (17 kDa, lane 5). Lane 2 contains the purification from suckerin produced in Saccharomyces cerevisiae. However, the protein was purified with a protocol optimized for bacteria. This could explain the smear.

We cooperated with the Miserez group from the Nanyang Technological University in Singapore. They have previously managed to produce suckerin-19 in E. coli with their plasmid pQE80-L SRT-19. They assisted us by sending their plasmid for further research. This plasmid contains suckerin-19 linked to a His-tag allowing for more precise purification using a Nickel-column. After purification, the samples were visualized on SDS-PAGE (Fig. 3). This allowed for comparison between suckerin-19 production via the pQE80-L SRT-19 strain and our own suckerin-8, -9, and -12 producing strains.

Figure 3. The SDS-PAGE gel demonstrates successful production and purification of suckerin-19.
Samples were loaded in duplo. Ladder (lane 1) and suckerin-19 (lane 2 and 3)

Constructing the optimal protein production plasmid

To achieve high protein production, easy protein purification and make an important contribution to the iGEM community, the iGEM Leiden team designed the iGEMized "ipET" plasmid (BBa_K3041014) for optimal recombinant protein production of RFC10 biobricks in Escherichia coli strains. Any protein gene can be added to this production plasmid and is automatically linked to a His-tag, enabling quick and easy His-tag purification of the desired protein after production. The construct allows for induced production of your desired protein by placing the gene under control of the T7/Lac promoter, thereby protein production is induced after the addition of IPTG. The ipET plasmid was obtained by adjusting the multiple cloning site (MCS) of pET28a(+) into the prefix and suffix, allowing for direct cloning of RFC10 biobricks into this high production plasmid (Fig. 4). More information about how this plasmid was designed can be found on our results page.

Figure 4. Schematic overview of the construction of ipET.
The replacement of the original multiple cloning site is displayed by an arrow leading the original MSC away from the construct. Then the RFC10 multiple cloning site is introduced using another arrow leading to the new construct. Doing this resulted in ipET.

The plasmid was validated by digestions. Three digestions using EcoRI, PstI and XbaI were performed, which are all newly added to the ipET plasmid. Linearization of the plasmid was observed in each digestion (Fig. 5).

Figure 5. Validation of ipET restriction sites
Lane 1 shows the undigested plasmid. lane 2 shows the plasmid linearized by XbaI digestion. In lane 3, ipET linearized by EcoRI digestion is shown. In the last lane, the plasmid linearized by PstI digestion is visualized.

Furthermore, the plasmid was sent to BaseClear for sequencing of the MCS region. The sequences showed strong alignment with the theoretical sequence suggesting the new multiple cloning site is correct (Fig. 6).

Figure 6. Sequence alignment of RFC10 multiple cloning site.
Two samples were sent for sequencing. Both showed a strong alignment with the multiple cloning site.

Unfortunately, due to time constraints, we have not been able to test this plasmid by introducing a biobrick. However, we still encourage future iGEM teams to use this plasmid, since validation showed the plasmid is correct. It is expected that this plasmid will increase protein production compared to standard iGEM plasmids and allows for accurate and quick purification methods.

References