Part:BBa_K4413008

pT7-RBS-R（PySp1）

PT7-RBS-R (PySp1) is a composite part containing PySp1

Introduction

The primary structure of PySp1 (R in brief) includes repeated sequences and N-terminal and C-terminal. The function of N-terminal and C-terminal is to promote the assembly of spider silk protein to form spider silk fiber and the function of the repeat region is mainly related to the mechanical properties of spider silk fiber. Therefore, we constructed an expression plasmid containing a repeat sequence, and expressed it in E. coli (BL21 strain), and then explored its physicochemical characteristics through subsequent characterization experiments

Experiment and Results

We obtained the repeat region sequence of protein PySp1 from the literature, and named it R in brief, and designed a prokaryotic expression system to express it. The composition part consist of T7 Promoter (BBa_I719005)、RBS (BBa_B0034)、R（BBa_K4413007）、T7 Terminator（BBa_K731721）（Figure 1). The T7 promoter controls the start of transcription, RBS sequence combines with ribosome to initiate the protein expression, and the 6X His sequence were inserted before the R for further protein purification. The target fragment was synthesized by a biological company, and then we recombined the R gene into plasmid with BamHⅠ and EcoRⅠ (Figure 2). In the end, we have successfully got the pET21a-R plasmid (Figure 3), and transformed it into E.coli (BL21 strain).

Figure1.Constitution of T7 Promoter-RBS-R-T7 Terminator gene circuits.

Figure 2：Ligation reaction of pET21a-R with T4 DNA ligase

Figure 3：Recombinant Plasmid Map of pET21a-R pET21 a vector contains Amp resistant fragments for screening positive clones

We verified the length of R and DNA sequence through PCR (Figure 4) and sequence alignment (Figure 5), The following results can prove that we have successfully constructed the prokaryotic expression system of R.

Figure 4：Electrophoresis of PCR products amplified from pET21a-R plasmid. M: DNA Marker；1 and 2：PCR results of positive clone; Product size: 906bp.

Figure 5：The results of Sequencing and Sequence alignments with expected sequence. a：Sequencing peak diagram.

Figure 5：The results of Sequencing and Sequence alignments with expected sequence. b：Sequence alignment between R and positive clone sequencing.

The R expression in E. coli was induced, and the bacterial solution was preliminarily verified by SDS-PAGE (Figure 6). After that, the target bands in the gel were cut off for mass spectrometry analysis (Figure 7).

Figure 6: Electrophoresis of pET21a-R with SDS-PAGE M：Marker；1：Before IPTG induction；2、3、4：After IPTG induction ；5、6：After purification

Figure 7a：The Mass Spectrometry of expressed Prorein R

Figure 7b：The Mass Spectrometry of expressed Prorein R

Finally, we purified the Protein R from the inclusion body with urea. Theoretically, this sequence closely related to the mechanical properties of spider silk fiber. In order to verify its function, we mixed the purified Protein R into the sodium alginate hydrogel (Figure 8). With or without Protein R as the control, the sodium alginate hydrogel exhibit different mechanical property. As shown in Figure 9 we tested the performance of the mixed hydrogel from the stretching, compression and puncture to explore the mechanic function of Protein R in the mixed hydrogel。We run the experiments with three replications and the data were recorded accordingly（Figure 9）.

Figure 8：Mixed hydrogel of Sodium alginate and Protein R C：4% Sodium alginate；T1：4% Sodium alginate+0.5% Protein R；T2：4% Sodium alginate+1%Protein R

Figure 9： Mechanic function test of mixed hydrogel

Conclusion

The above results indicate that we have successfully constructed the prokaryotic expression system of R protein and obtained R protein by IPTG inducing. In addition, we tested the mechanic properties of Protein R combined with sodium alginate hydrogel, which could be applied as raw materials widely used in 3D printing. And we found that Protein R has an obvious effect on the resistance in stretching and puncture，while its resistance to compression is relatively weaker.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 711
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 387
  Illegal BglII site found at 402
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]