Engineering Success

Our project had many challenging aspects, and many of these required their own engineering cycles. However, our work on the major strain spider silk is a great representative of our work with a common challenge in our project: difficult inserts.

Major Strain Spider Silk: From Insert to Plasmid

Background

Spider Silk is one of the three biopolymers used as components of our bricks. Spiders produce seven different versions of Spider Silk in their nets. For our biopolymer we chose the “major strain spider silk (MaSp)” (Fig.1). This version of spider silk is very robust and therefore used to enhance the stability of our bricks. During this project, three different plasmids containing the major spider silk gene were developed to receive information as to which type of plasmid ensures the highest expression rates.

Fig. 1: Different types of Spider Silk (Source: Eisoldt et al., 2011)

The Insert

Design

Major strain spider silk consists of four major parts, the first T-sequence, two S-sequences and another T-sequence at the end (Fig. 2). Due to a high number of repeats in the sequence, synthesising the whole sequence is not possible in one try. The engineering design was therefore split into two steps, allowing us to order the insert in smaller parts, which needed to be combined to gain the whole sequence for the major strain spider silk.

Step 1: In a first step, only the T-sequence in the beginning and the first of the two S-sequences were created using polymerase chain reaction (PCR)(Fig. 3). The S-sequence could be ordered at the company IDT, when split into three different parts (S#1, S#2 and S#3). The T-sequence on the other hand could not be ordered at the company IDT. Thus, various primers, resulting in the T-sequence when combined, were ordered at the company IDT.

In a first trial run a PCR was done containing all the components for part 1 of the engineering design. However, no band in the expected size could be seen on the electrophoresis performed after the PCR.

After discussing these results and brainstorming solutions, sequence-T, Part S#1, Part S#2 and Part S#3 were amplified by PCR separately. The reaction resulting in the T-sequence consisted of the primers “FW1 A1T”, “BW1 A1T”, “FW2 A1T” and “BW2 A1T”. For the parts S#1, S#2 and S#3 the template sequences were mixed with the according primers resulting in the three following reactions. Template “A1S#1” and primers “FW2 A1T” and “A1Spart1and2BW” for the part S#1, template “A1S#2” and primers “A1Spart1and2FW” and “A1Spart2and3BW” for the part S#2 as well as template “A1S#3” and primers “A1Spart2and3FW” and “A1Spart3BW”. To combine these products in a second reaction, the primers “FW1 A1T”, “BW2 A1T”, “A1Spart1and2FW”, “A1Spart1and2BW”, “A1Spart2and3FW”, “A1Spart2and3BW”, and “A1Spart3BW” were used. The success of this experiment was proven by sequencing the cleaned PCR product at the company “Microsynth”.

Step 2: The PCR product of step one was now amplified with three different sets of primers to gain the parts A, B and C together creating the insert used in the following Golden Gate cloning to create the designed plasmids. These primers also contained recognition sites for the restriction enzyme BsaI, resulting in sticky ends with the ability to combine the PCR products to the correct sequence, as well as the possibility to add the secretion factor and clone the whole insert into a backbone 1 with the fusion sites 2 and 3.

Part A was constructed with the use of the primers “A1T FW1” on the on hand and “PCR-2-BW” on the other hand (Fig. 4), resulting in a N-terminal sticky end with the overhang sequence “GAGA”, fitting the C-terminal sticky end of the secretion factor for Pichia pastoris, and a C-terminal sticky end with the overhang sequence “CGCT”, fitting the N-terminal sticky end of part B. This part consists of the N-terminal T-sequence as well as the first of the two S-sequences.

For part B the primers “PCR-3-FW” and “PCR-3-BW” were used for the creation of a PCR-product (Fig.5) containing only one S-sequence including the N-terminal sticky end with the overhang sequence “CGCT”, fitting the C-terminal sticky end of part A and the C-terminal sticky end with the overhang sequence “GGCG”, fitting the N-terminal sticky end of part C. Part B is used as the second of the two S-sequences in the complete insert.

The primers “PCR-4-FW” and “PCR-4-BW” were necessary to produce the small PCR product C, only including the C-terminal T-sequence (Fig. 6). This PCR product contains the C-terminal fusion site 3, needed for the cloning in backbone 1, as well as the N-terminal sticky end with the overhang sequence “GGCG”, fitting the C-terminal sticky end of part B.

The resulting parts A, B and C were determined to contain the expected sequences by sending them to the company “Mikrosynth” for sequencing, after cleaning up the PCR-products.

Golden Gate Cloning

Backbone 1

The cloning in backbone 1 (Fig. 7)combines the three parts of the main strain spider silk sequence with the secretion signal for Pichia pastoris. This secretion signal contains the N-terminal fusion site 2, used for cloning into backbone 1 and the C-terminal sticky end with the overhang sequence “GAGA”, fitting the N-terminal sticky end of part A, when cut with the restriction enzyme BsaI. To later gain inside on the success of the Golden Gate reaction, the backbone 1 contains a kanamycine resistance gene.

After the Golden Gate reaction, the resulting plasmid was transformed into chemically competent E.coli of the strain DH10b by heatshock treatment. For the determination of a successful reaction a negative control was added to the Golden Gate reaction. After plating the transformed E.coli on LB-media plates containing the antibiotic kanamycine and incubating over night at 37 degrees Celsius, cell growth could only be witnessed on the plates containing the E.coli with the correct plasmid. However, after extracting the plasmid, using the “MiniPrep” method, choosing the samples to send for sequencing by doing a restriction digest to get slight knowledge about the composition of the plasmid and sending the chosen samples for sequencing, slight mutations in the coding sequence could be noticed in all the samples.

Since only a few mutations occurred and the sequencing results of the PCR products clearly showed the expected sequences, the decision to just repeat the Golden Gate experiment was made. The second run proved to be very successful as every sample sent to sequencing matched the expected sequence.

Backbone 2

Backbone 2 was only needed for the plasmid containing the four coding sequences, since it can otherwise be skipped by using by using an empty backbone 3 with the fusion sites 1 and 4, created by digest with BbsI.

For the plasmid version including the coding sequence for main strain spider silk four times, four different backbone 2 plasmids were made. To avoid problems caused by inadequate binding of DNA in repeating regions, two promoters and three terminators were used in the four backbone 2 plasmids.

Every backbone 2, used for the main strain spider silk contains the fusion sites 1 and 4, created by digest with BbsI, as well as an ampicillin resistance used for the determination of a successful cloning. The used promoters contain the N-terminal fusion site 1 and the C-terminal fusion site 2, fitting the N-terminal fusion site 2 of the secretion factor, whereas the used terminators contain the N-terminal fusion site 3, fitting the C-terminal fusion site 3 of the main strain spider silk and the C-terminal fusion site 4, used for cloning in backbone 2, when digested with the restriction enzyme BbsI.

The difference between the used backbones lies in the fusion sites, created by a digest with BsaI. This digest is used for the cloning in backbone 3.

To assemble the backbone 2-versions, a Golden Gate reaction with the restriction enzyme BbsI, was performed. The reactions entailed the different promoters and terminators as well as the previously created backbone 1, containing the major strain spider silk insert including the secretion signal for Pichia pastoris.

The first created backbone 2 consists of the promoter “pGAP”, the terminator “RPL2Att”, the MaSp insert including the secretion signal and a backbone 2 version building the fusion sites A and B after a digest with the restriction enzyme BsaI. The N-terminal fusion site A is used for the cloning in backbone 3 while the C-terminal fusion site B fits the N-terminal fusion site B of the second created backbone 2.

For the second backbone 2-version the promoter “pTEF”, the terminator “RPP1Btt” and the MaSp insert including the secretion signal were cloned in an empty backbone 2, building the N-terminal fusion site B, fitting the C-terminal fusion site B of the first backbone 2 and the C-terminal fusion site C, fitting the N-terminal fusion site C of the third backbone 2 version, when digested with the restriction enzyme BsaI.

The third created backbone 2 consists of the promoter “pGAP”, the terminator “RPS25Att”, the MaSp insert including the secretion signal and a backbone 2 version building the fusion sites C and E after a digest with the restriction enzyme BsaI. The N-terminal fusion site C fits the C-terminal fusion site C of the second backbone 2 while the C-terminal fusion site E fits the N-terminal fusion site B of the fourth created backbone 2.

For the fourth backbone 2-version the promoter “pTEF”, the terminator “RPP1Btt” and the MaSp insert including the secretion signal were cloned in an empty backbone 2, building the N-terminal fusion site C, fitting the C-terminal fusion site C of the third backbone 2 and the C-terminal fusion site E, used for cloning in a backbone 3 with the fusion sites A and E, when digested with the restriction enzyme BsaI.

The Golden Gate products of the four backbone 2 versions were transformed into chemically competent E.coli DH10b and plated on LB-Ampicillin Agar plates. After an incubation period of 18 hours at 37°C, colony growth could only be witnessed on the plates containing the complete backbone 2 versions, but not on the control plates only containing the empty and, after the Golden Gate, linearized backbone 2 versions. As further proof for the successful cloning a control digest was performed using the restriction enzyme BsaI. After loading the digest on a 1% agarose gel, the bands for all backbone versions could be found at the expected sizes.

Backbone 3

To obtain a comparison regarding expression efficiency, three different backbone 3 were created. The first and second version each contain one expression cassette that varies only in terms of the used promoter. The third expression cassette entails four different expression cassettes.

The backbone 3 were obtained by Golden Gate cloning. All backbone three versions contain a nourseothricin resistance to enable a selection of the E.coli clones including the plasmid after the Golden Gate reaction was transformed into E.coli.

BB3-pGAP-MaSp-RPL2Att

The first created backbone 3 is the plasmid BB3-pGAP-MaSp-RPL2Att (Fig.8). It contains the promoter “pGAP”, the terminator “RPL2Att” and the MaSp insert including the secretion signal. This plasmid was synthesized by doing a Golden Gate reaction including the Backbone 1 with the MaSp coding sequence and the secretion signal for Pichia pastoris, the “pGAP” promoter and the “RPL2Att terminator. The restriction enzyme BbsI was used to create the desired sticky ends. For the empty backbone the backbone 3 version, building the fusion sites 1 and 4, when digested with BbsI, was chosen.

After an incubation period of 18 hours at 37 degrees Celsius, colony growth could only be witnessed on the nourseothricin-plates containing the complete BB3-pGAP-MaSp-RPL2Att plasmid but not on the control plates only containing the empty and, after the Golden Gate, linearized backbone 3. As further proof for the successful cloning a control digest was performed using the restriction enzyme BglI. After loading the digest on a 1% agarose gel, the bands could be found at the expected sizes.

BB3-pTEF-MaSp-RPL2Att

The first created backbone 3 is the plasmid BB3-pTEF-MaSp-RPL2Att (Fig. 9). It contains the promoter “pTEF”, the terminator “RPL2Att” and the MaSp insert including the secretion signal. This plasmid was synthesized by doing a Golden Gate reaction including the Backbone 1 with the MaSp coding sequence and the secretion signal for Pichia pastoris, the “pTEF” promoter and the “RPL2Att terminator. The restriction enzyme BbsI was used to create the desired sticky ends. For the empty backbone the backbone 3 version, building the fusion sites 1 and 4, when digested with BbsI, was chosen.

After an incubation period of 18 hours at 37 degrees Celsius, colony growth could only be witnessed on the nourseothricin-plates containing the complete BB3-pTEF-MaSp-RPL2Att plasmid but not on the control plates only containing the empty and, after the Golden Gate, linearized backbone 3. As further proof for the successful cloning a control digest was performed using the restriction enzyme BglI. After loading the digest on a 1% agarose gel, the bands could be found at the expected sizes.

BB3-4xMaSp

The third plasmid version was created out of the four previously described backbone 2 versions. These backbone 2 versions were used in a Golden Gate reaction with BsaI as restriction enzyme, also including an empty backbone 3 containing the fusion sites A and E after digest with BsaI.

After an incubation period of 18 hours at 37°C, extensive colony growth could be witnessed on the nourseothricin-plates, whereas only few colonies appeared on the plates only containing the empty and, after the Golden Gate, linearized backbone 3. A first control digest with the enzyme BglI was not entirely rewarding since it led to several not very distinctive bands. After some consideration a different restriction enzyme, XhoI, was chosen for a second digest. The second digest was more successful, resulting in bands at the expected sizes on a 1% agarose gel.

Expression in Pichia pastoris

To transform the obtained plasmids into the genomic DNA of Pichia pastoris, they were first linearized using the restriction enzyme AscI. The restriction site of AscI is located between the two pRGI parts of the plasmid, later attaching to homologous parts in the genomic DNA, while inserting the MaSp expression cassette.

The transformation was done by electroporation into electrocompetent Pichia pastoris (strain CBS2612). The transformed Pichia pastoris was plated on YPD-Agar plates containing nourseothricin and incubated for 72 hours at 28°C. After the incubation period, a large number of colonies could be noticed on the plates containing the plasmids compared to only a few colonies on the negative control, transformed only with sterile water.

Four colonies of each transformed Pichia pastoris were chosen and plated on new YPD-Agar plates containing nourseothricin, using the fractional smear method. After an incubation period of 72 hours at 28°C, this step was repeated with colonies of the new plates, ensuring the purity of the clone.

A preculture of 10mL of liquid YPG-medium containing nourseothricin was inoculated with two clones for each plasmid transformed in Pichia pastoris and incubated for at 28°C for 24 hours shaking at 180 RPM. After the incubation period a main culture of 50mL liquid BM-Medium with a glucose concentration of 2% was set to an OD600 of 0,1 and incubated for 48 hours. After 12, 24, 36, 48 hours, 2mL samples were taken and the glucose concentration was set to 1%.

1mL of the obtained samples was centrifuged at 16,000xg for 5min. The supernatant was used as a sample for SDS-Page, confirming the expression of the main strain spider silk protein. The other 1mL was used to monitor the cell growth by OD600 measurement.

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.11). 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.