1) Antibiotics
2) hemolymph
3) Mcl1 promoter
We designed the primers PMcl1-f(ACGTC//CTGCAG//AATCATGCAGCGCTATGAG, with a PstI site underlined) and PMcl1-r(ATAA//GCGGCCGC//CATGATGGTCTAGGGAACG with a NotI site underlined), according to the PMcl1 sequence[1], to amplify the Mcl1 promoter region with Mcl1 mRNA 5'-untranslated region at the 5' end of the coding region. The whole length is 2772bp.
The gel image below shows that we succeed extracting the Mcl1 promoter and its 5'-untranslated region (99bp downstream the promoter) from the genomic DNA of our chassis organism M. anisopliae ARSEF549.
Then we digested the DNA fragment with NotI and PstI in order to insert it into the backbone. However, when we ran gel electrophoresis to check the digestion result, we found that there is still one unknown PstI cut site inside the PMcl1 region.
We decided to sequence this DNA fragment we extracted and mutate the PstI site, but we didn't have enough time to finish our relative vectors construction.
4) KillerRed expression in M.anisopliae
We constructed a KillerRed expression cassette with a fungal promoter PgpdA and a fungal terminator TtrpC. This cassette was used to confirm that KillerRed can be expressed in M.anisopliae
*The following fluorescence images indicated that KillerRed was successfully expressed in M.anisopliae.
As we observed, the growth situations of M.anisopliae KR transformants on media will not be affected greatly since irradiation of KillerRed localized in cell cytosol has a weak effect on cell survival in eukaryotic cells. In advance, we decided to fuse a SV40 NLS to the KillerRed protein( http://parts.igem.org/Part:BBa_K2040122 BBa_K2040122 ) so that KillerRed can function in the ROS-sensitive intracellular localizations, the chromatin in nucleus, due to the NLS.
References:
[1]
