Part:BBa_K4335003

ChlamyHgR

We used the Rbcs2 promoter and terminator, and since the Rbcs2 Promotor Rhizobium-derived transcription factor was low, we added the UTR of the heat shock promoter HSP70A Promotor to enhance the promoter and thus the heterologous expression of the Staygold fluorescent protein StayGold, a green fluorescent protein (GFP) from the jellyfish family Endocystis, was chosen as our reporter fluorescent protein because it is orders of magnitude more photostable than any currently available fluorescent protein, and because the excitation and emission wavelengths of Staygold do not overlap with the chloroplast fluorescence of Chlamydomonas reinhardtii itself.

Usage

We inserted ChlamyHgR into plasmids pTX2038 and pTX2040 as marker genes for transformation screening.

Result

Plasmid construction

To verify our successful assembly, we designed two primers, Hgy-F and Hgy-R, for PCR verification. The primer location, primer sequence and gel map are shown in the following figure:

mCherry and the position of primer targeting

Electrophoregram of amplification products,M is DNA Marker.

Verification of transformation results

We introduced plasmids pTX2038 and pTX2040 containing APHVII ChlamyHgR gene into Chlamydomonas reinhardtii by electrical transformation.

We used the primer with a length of about 600 bp designed according to the Hyg resistance gene to carry out PCR validation on the single colonies successfully transferred into pTX2038 and ptx2040.

Figure 4. Sequence alignment of Hyg resistance gene.
M:2000bp DNA marker; WT:wild type; Plasmid: Linear plasmid corresponding to the vector.

Figure 4. shows that the DNA sequence in the single algal colony was successfully amplified by PCR from pTX2038 and pTX2040. From this electrophoretogram, we can see the brightness of Hyg resistance gene fragment is consistent with the DNA bands of the positive control group, and match with the DNA Marker marking position, which indicates that the PCR products of the fragments transferred into the vector are in a high concentration and normal expression state.

Reference

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 782
  Illegal NgoMIV site found at 890
  
• 1000
  COMPATIBLE WITH RFC[1000]