Engineering

Cycle 1: Ubx

Design

To obtain insights associated with Ubx protein properties, we created a composite part K4377006. (Figure 1). First, we attached 10x His tag at the N terminal of Ubx in order to purify the protein after expression. The attachment of 10x His tag on N terminal instead of C terminal is for the purpose to further amplify out production of Ubx. Second, we performed codon harmonization to reduce protein incorporation in expressed bacteria. The optimized sequence of codons is expected to demonstrate better expression by our engineered E. coli.

Build

Cloning result

We successfully amplified Ubx plasmid in E. coli DH5α and transformed into E. coli BL21 for expression.

SDS-PAGE result

After expression, we conducted SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) to verify whether the protein was produced (result in Figure 3).

Test

As shown in the SDS-PAGE result, the production was below expectation. We further conducted functional tests, but there was no significant evidence that the protein is able to form dityrosine bonds.

Learn

After discussion, we decided to focus on finding approaches to achieve better production. Thus, we designed engineering Cycle 2.

Next Cycle

Cycle 2: Binding Region Prediction

Design

In cycle 2, we developed approaches to narrow down to dityrosine containing domains among Ubx sequence, as dityrosine cross-linkings are evidenced to drive the assembly of proteins and peptides.4 In step 1, we used the ANCHOR algorithm to predict the region among Ubx sequences that demonstrate better binding. On the other hand, a paper suggested that the position of lysine will affect the rate of formation of dityrosine bonds.5 This has further led us to our step 2 - building a model to precisely predict exact formation of dityrosine cross linking among the possible binding sites.

Build

After the analytics of the ANCHOR algorithm in step 1, we further utilize the results and build a selection model to analyze the distance of lysine and tyrosine. The selection model has further specified the binding probability of tyrosine. Based on the step-wise manner of our design, by inputting a baseline value of binding possibility and the sequence to be analyzed, the model will output a line chart based on the list below.

Test

Learn

According to the result of the ANCHOR algorithm and the model we built, Y167 region and Y240 of the Ubx showed the best probability of forming dityrosine cross linking.6 Thus, we further engineer Y167 and Y240 in our cycle 3.

Next Cycle

Cycle 3: Y167 mRFP Y240

Design

With the result of cycle 2, we chose to further design Y167 and Y240 with mRFP in our cycle 3. We first engineered functional protein mRFP (Part:E1010) with Y167 Y240 short peptides in the attempt to verify the self-assembly function. We chose mRFP because it is easy to observe. Second, we attached 10x His tag at the N terminal of Ubx protein in order to purify our protein after expression. The reason we add 10x His tag at N terminal but not C terminal is to amplify the production of Ubx protein. Lastly, we performed codon harmonization to reduce protein incorporation in expressed bacteria. The optimized sequence of codons is expected to demonstrate better expression by our engineered E. coli.

Build

Cloning result

With the same process of building Composite part K4377006, we successfully built Composite part K4377009.

SDS-PAGE result

After expression, we conducted SDS-PAGE to verify the protein was produced (result in Figure 8).

Test

Solubility of Y167 mRFP Y240

The pellet of Y167 mRFP Y240 plasmid remained red after ultrasonic disruption and centrifuged in PBS buffer. It is assumed that Y167 mRFP Y240 might be insoluble. Therefore, we conducted SDS-PAGE to the amount of Y167 mRFP Y240 in the pellet and supernatant respectively.

The result showed the pellet contains more amount of Y167 mRFP Y240 than supernatant in the PBS buffer. In order to obtain more amount of Y167 mRFP Y240, we ultrasonic disrupted Y167 mRFP Y240 pellet and control (pSB1A3 plasmid pellet) in 8M Urea (high concentration chaotropic agent). After disruption, we centrifuged the sample and separated the supernatant to dialyze into Tris Base buffer and ran SDS-PAGE (result in Figure 10).

Also, we measured the mRFP intensity of the supernatant in both PBS and Tris Base buffer (result in Figure 11).

As Figure 11 shows, mRFP intensity of the supernatant in Tris Base buffer is way higher than the one in PBS buffer. The result shows that mRFP (as functional protein) remains its function after it is fused with two short peptide areas. Then, we conducted the same process for further experiments.

Functional Test

To prove that our protein crosslinks with dityrosine bonds. We confirmed the formation of dityrosine bonds from FT/IR and CD measurements of our sample. Later, we explored the application of our biomaterial. Viscosity test results showed that our sample has potential to be bioink. We later 3D printed our sample and acquired the ideal outcome.

We used FT/IR-4700 to analyze additional and reduced bondings before and after processing with EDTA-Fe and H2O2. Data proves crosslinking reaction came from dityrosine bond formation with the obvious decrease in C-H bonds. (result in Figure13.)

We did measurement of viscosity for our protein sample and confirmed its shear thinning behavior, which shows high potential for the future development of bioinks for 3D printing.

Learn

Through functional tests, we had successfully achieved our goal of creating the biomaterial that can crosslink and preserve the function of functional protein fusion with them. It is concluded that our biomaterial has the potential to successfully incorporate diverse functional proteins for wide applications in various fields.