HDLEA1 with strong promoter/RBS
A strong constitutive promoter (J23100) and strong ribosome binding site (B0034) have been placed upstream of the HDLEA1 in order to maximize expression of the protein to provide the ability to beter survive desiccation circumstances.
Part BBa_K2128204 (the underlying Coding Sequence in part BBa_K2128004) was validated using a desiccation protocol that demonstrated the part worked as expected (i.e., improved survivability when the HDLEA1 coding sequence was expressed compared to when it was not expressed). The K2128204 part consists of the Biobrick part K880005 (i.e., a strong constitutive promoter J23100 and strong ribosome binding site B0034) followed by the HDLEA1 coding sequence from K2128004. When placed on the high-copy-number plasmid backbone pSB1C3, maximal expression of HDLEA1 is expected. The comparative reference was K880005, also on pSB1C3 backbone (i.e., the plasmid under test minus the HDLEA1 coding sequence).
The desiccation protocol was initially run to identify serial dilution values for each hour of desiccation that would result in a number of colony forming units per plate (CFU/plate) on the order of 30-300. The OD600 of the initial overnight culture was taken (using a 1:10 dilution proxy) in order to obtain the expected number of CFUs in 20uL using the formula (0.02mL)*(8x108 CFU/mL/OD600)*OD600. Excellent agreement (within ±25%) with the observed average CFU/plate was found for both the reference and test plasmid systems after correcting for the dilution factor for H=0 hours of desiccation (i.e., no desiccation).
For each hour (H=0, 4, 7.5 and 22.5) and each plasmid system under test (i.e., the K880005 reference and the HDLEA1-based K2128204), data were collected in triplicate and used to obtain an average and standard deviation for CFU/plate. Plates with evidence of contamination or pipetting error were removed (a total of two plates out of 24). The dilution factor was corrected for in order to obtain the CFU in the desiccated 20uL aliquot and normalized by the measured CFU in a non-desiccated 20uL aliquot to obtain a survival rate versus time (i.e., fraction of surviving colonies). The results are shown in Figure 1 below.
As expected, the fraction of surviving cells decays monotonically with increasing desiccation time for both the reference (“REF”) and HDLEA1-based systems (“TEST_HDLEA1”). Beyond four hours, the plasmid expressing HDLEA1 shows an improved ability to survive desiccation in a manner that is statistically significant based on measured standard deviations. In particular, the amount improvement is estimated to be 3.2-fold (±1.5) for H=7 hours of desiccation and 2.5-fold (±1.3) for H=22.5 hours of desiccation. This approximately three-fold improvement in survivability validates that the part works as expected.
Sequence and Features
- 10COMPATIBLE WITH RFC
- 12INCOMPATIBLE WITH RFCIllegal NheI site found at 7
Illegal NheI site found at 30
- 21COMPATIBLE WITH RFC
- 23COMPATIBLE WITH RFC
- 25COMPATIBLE WITH RFC
- 1000COMPATIBLE WITH RFC