Part:BBa_K4413009

pT7-RBS-EGFP-R（PySp1）

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

Introduction

BBa_K4413008 contains RBS and R genes. We plan to improve this cassette, and constructed the improve part (BBa_K4413009). We inserted an EGFP sequence in the upstream of R. In addition, we located the expression position of EGFP-R fusion protein in E. coli through fluorescence microscope, and obtained the hydrogel mixed with EGFP-R fusion protein.

Experiment and Results

We designed a prokaryotic expression system with fluorescent tags, inserting the EGFP sequence upstream of the R to make the protein show green fluorescence. The composition part consist of pT7(BBa_I719005),RBS(BBa_B0034),EGFP(BBa_S03452) and R（BBa_K4413007）（Figure 1).The EGFP fragment is cloned from the plasmid by PCR, and the 5', end of the primer used in PCR contain the upstream and downstream homology arms of the pET21a gene respectively，the PCR fragment size is 1395 bp (Figure 2). Integration of EGFP fragments and linearized vectors pET21a through homologous recombination (Figure 3), Finally, we successfully recombined the pET21a-EGFP-R plasmid (Figure 4), and transferred it into E. coli (BL21) strain.

Figure 1： Constitution of T7 Promoter-RBS-EGFP-R-T7 Terminator gene circuits.

Figure 2：Electrophoresis of PCR products amplified from plasmid. M: Marker; 1、2: EGFP（720bp）

Figure 3：Homological recombination of pET21a-EGFP-R Vector：Linearized vector pET21a-R；Fragment：EGFP

Figure 4：Recombinant Plasmid Map of pET21a-EGFP-R. pET21a vector contains Amp resistant fragments for screening positive clones

We verified the length of EGFP-R sequence by PCR (Figure 5), and induced the BL21 strain by IPTG , Then we can observed obvious green fluorescence with fluorescence microscope.

Figure 5：Electrophoresis of PCR products amplified from pET21a-EGFP-R plasmid.M: Marker; 1、2: PCR results of positive clone; Product size (EGFP-R): 1600bp

EGFP is an excellent reporter gene, which can mark and locate the fusion protein. Through fluorescence microscope, we can accurately locate the expression position of protein R (Figure 6), and we mix the purified protein with sodium alginate hydrogel (Figure 7), making our product more intuitive and convenient for positioning in subsequent experiments.

Figure 6:E.coli cells under fluorescence microscope a：E.coli with R；b：E.coli with EGFP-R .

Figure 7：Hydrogel with EGFP-R and normal R under blue light a:Mixed hydrogel with Sodium alginate and Protein EGFP-R b:Mixed hydrogel with Sodium alginate and Protein R.

Conclusion

The above experimental results show that we have successfully constructed the prokaryotic expression vector of EGFP-R fusion protein. Compared with the original part, the fusion protein can be directly observed by fluorescence microscope or even eyes. Thus, we can now both increase the mechanic properties of the hydrogel, and locate our printed biomaterials for further tracing and maintenance.

Reference

Blasingame E, Tuton-Blasingame T, Larkin L, Falick AM, Zhao L, Fong J, Vaidyanathan V, Visperas A, Geurts P, Hu X, La Mattina C, Vierra C. Pyriform spidroin 1, a novel member of the silk gene family that anchors dragline silk fibers in attachment discs of the black widow spider, Latrodectus hesperus. J Biol Chem. 2009 Oct 16;284(42):29097-108. doi: 10.1074/jbc.M109.021378. Epub 2009 Aug 7. PMID: 19666476; PMCID: PMC2781455.

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 1437
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 1113
  Illegal BglII site found at 1128
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]