Team:Genspace/Measurement
Desiccation Test Results for HDLEA1 (K2128100)
Part K2128204 (and the underlying Coding Sequence in part 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 the figure 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.
qPCR Results
A preliminary experiment was run using lysate from 1 million cells per reaction. Eight replicates were run for each sample type (with or without the reporter). After excluding outliers caused by edge effects, average CT values for six replicates for each sample were compared:
Test: 17.02
Control: 21.48
Assuming a perfect doubling of product for each CT, this would indicate 22 times as many copies of the target sequence in the test samples compared to the control, or a PCN of ~21 copies per cell.
While this result was an order of magnitude less than the expected value, the consistency among replicates of each sample encouraged us to move forward with absolute quantification experiments.
pSB1C3 absolute quantification run #1
Lysate from 1 million stationary phase cells harboring K909006-pSB1C3 was run against a 3-point standard of 106, 107, and 108 copies.
pSB1C3 absolute quantification run #2
Lysate from 100,000 mid-log phase cells harboring K909006-pSB1C3 was compared against a 3-point standard of 105, 106, and 107 copies. Due to the reduced amplification efficiency of the 108-copy standard in run 1, cell numbers were reduced 10-fold in all subsequent experiments.
Note: The K909006-pSB1C3 harboring cells used for this run were lysed in mid-log phase, which may account for the reduced PCN.
pSB1C3 absolute quantification run #3
Lysate from 100,000 stationary phase cells harboring K909006-pSB1C3 was compared against a 3-point standard of 105, 106, and 107 copies.
pSB1C3 absolute quantification run #4
Lysate from 100,000 stationary phase cells harboring K909006-pSB1C3 was compared against a 2-point standard of 105 and 106 1.1x106 copies. The 1.1x106-copy standard was created using lysate from 105 cells as well as 106 copies of purified plasmid. This point was created to test for variance in amplification efficiency of plasmid vs. genomic template.
qPCR Conclusions
Due to time constraints, only a few qPCR runs could be completed prior to the Wiki freeze, so this data is still preliminary.
