Part:BBa_K584026
Constitutive_Promoter + AFP(Antifreeze extra-cellular Protein Generator)
This part contains the constitutive promoter (J23119) and the Antifreeze Protein extracellular protein generator (BBa_K584020) sequence behind it.
The part BBa_K584020 contains OmpA_transmembrane_domain + Linker + point_mutant_HPLC6(AFP) with the B0034 RBS and B0015 Terminator.
A point mutation at Base Pair number 156 of the HPLC6 sequence was performed by replacing the T in the wild-type coding sequence to C. This mutation was necessary as the sequence CTGCAG in the Wild-type coding sequence corresponded to a Pst1 restriction site. GCT(Wild type) and GCC(1 point mutant) are both valid triplet codons for Alanine.
Detailed information of the Pseudopleuronectes americanus Antifreeze Protein (HPLC gene) can be found on this NCBI [http://www.ncbi.nlm.nih.gov/nuccore/M62417 webpage].
Characterization
Note: to generate supercooled water, ultrapure “milli-Q” water (Millipore Corporation) was filtered over a 0.22µm filter to generate water devoid of nucleating species. All the experiments were done in TOP10F' cells.
As a negative control, TOP10F’ cells constitutively expressing GFP (brick BBa_K584001) were used.
An overnight preculture was inoculated at an initial OD of 0.1 in 15mL medium, and grown for 5 hours at 37°C. Cells were spun down and resuspended in 1 mL filtered water, after which they were washed twice with 1mL filtered water. The ODs of the resulting cell suspensions were determined and all cells were diluted to the same OD of 10.
In the meanwhile, the ethanol bath was set at -10°C, and clean, plastic tubes filled with 5mL filtered water were put in the chilled bath to create supercooled water at -10°C.
For the AFP test, the same amount of AFP-expressing or control cells were added to the 5mL supercooled water and checked for freezing/antifreeze activity (see videos).
As can be seen on the videos, addition of cells expressing AFP as well as control cells induced the freezing of the water at -10°C (due to aspecific nucleation of the cells), so no antifreeze activity could be demonstrated under this condition. To make final conclusion on the antifreeze activity of our biobrick, we want to test additional temperatures between -10°C and -6°C (at which control cells do not induce freezing, see characterization of brick BBa_K584027). If we can find a specific temperature at which the control cells would induce freezing, while AFP-expressing cells would not, we would demonstrate antifreeze activity of our brick. However, due to time constraints of the Wiki freeze, we could not perform these experiments yet.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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