Part:BBa_K4765126

ribozyme connected: H. ex mtSSB + SAHS 33020 + AnAFP

Introduction

This composite part combines BBa_K4765016(H. ex mtSSB),BBa_K2306003(SAHS 33020)and BBa_K4765015(AnAFP) in our ribozyme-assisted polycistronic co-expression system：pRAP.

AnAFP is an antifreeze protein that endows E. coli with antifreeze capability. SAHS 33020 is a heat-soluble protein found in the tardigrades which increases E. coli 's stability against desiccation. H. ex mtSSB is a type of mitochondrial single-stranded DNA binding protein that improves E. coli 's resistance to desiccation and UV radiation.

Characterization

Agarose gel electrophoresis

Figure 1. Agarose gel electrophoresis of PCR products, amplified from bacterial colonies/cultures. From left lane(1) to right lane(3) indicate the successful construction of H. ex mtSSB, H. ex mtSSB + SAHS 33020, and H. ex mtSSB + SAHS 33020 + AnAFP.

Desiccation Survival Assay

We constructed H. ex mtSSB, SAHS 33020, and AnAFP into this part using pRAP. Within BBa_K4765127, we rearranged the positions of these three proteins.

Figure 2. Workflow of Desiccation Survival Assay

We've designed an experimental group with E. coli expressing H. ex mtSSB, SAHS 33020, and AnAFP, and used E. coli transformed with the empty pET28a vector as a control. All proteins are expressed via leakage in E. coli BL21 DE3. All the groups are incubated overnight. After reaching an OD600 value of 1.0, the liquid culture is centrifuged, and the supernatant is removed. Pellets are dried for 6.5 hr in SpeedVac (Savant SpeedVac SC100) under 4 0 °C. Finally, the pellets are weighed and resuspended in LB medium and dilute 10^5-fold for CFU counting.

Through experiments, we observed that when both H. ex mtSSB and SAHS 33020 are co-expressed, E. coli exhibits a higher survival rate under desiccation compared to E. coli expressing these two proteins individually. Furthermore, altering the order of these three proteins in pRAP does not affect the survival rate of E. coli, indicating that pRAP can correctly express these proteins, regardless of their position.

Figure 3. The dry weight of all the groups after drying There is no statistically significant difference in the dry weights among the experimental groups, indicating that the number of E. coli is consistent between the bacterial tubes.

Figure 4. CFU colony count of all the groups after drying The E. coli expressing H. ex mtSSB, SAHS 33020, and AnAFP together showed higher CFU counts after drying compared to E.coli expressing H. ex mtSSB and SAHS33020 individually. Furthermore, there's no significant difference between the two E. colis with rearranged CDS positions. Mean values from three rounds of independent experiments are shown. Huge error bars suggest variations between rounds of experiments.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 247
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 880
  Illegal BsaI.rc site found at 515
  Illegal BsaI.rc site found at 596
  Illegal SapI site found at 409
  Illegal SapI.rc site found at 1804