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

Characterization

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.

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, 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 1. The dry weight of all the gourps after freeze-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 2. CFU colony count of all the groups after freeze-drying CFU colonies/mL= CFU count*10^6 The E. coli expressing H.ex mtSSB, SAHS 33020 and AnAFP together showed higher CFU counts after freeze-drying compared to E.coli expresssing H. ex mtSSB and SAHS33020 individually. Futhermore there's no significant difference between the two E. coli with rearranged protein positions.

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