Difference between revisions of "Part:BBa K4806212"
| Line 16: | Line 16: | ||
<h2>Construct</h2> | <h2>Construct</h2> | ||
<p> | <p> | ||
| − | <img class="bild" src="https://static.igem.wiki/teams/4806/wiki/registry/level2/ | + | <img class="bild" src="https://static.igem.wiki/teams/4806/wiki/registry/level2/por-ctppsad-construct.png"> |
<div class="unterschrift"><b>Fig.1 Construct design</b><br> | <div class="unterschrift"><b>Fig.1 Construct design</b><br> | ||
This construct was designed using the modular cloning system (MoClo).</div> | This construct was designed using the modular cloning system (MoClo).</div> | ||
| Line 35: | Line 35: | ||
<p>We confirmed that this construct is built correctly via agarose gel electrophoresis.</p> | <p>We confirmed that this construct is built correctly via agarose gel electrophoresis.</p> | ||
<p> | <p> | ||
| − | <img class="agarose" src="https://static.igem.wiki/teams/4806/wiki/registry/level2/ | + | <img class="agarose" src="https://static.igem.wiki/teams/4806/wiki/registry/level2/por-ctppsad-agarose.png"> |
<div class="unterschrift"><b>Fig.2 Test digest of the POR level 2 targeted to the chloroplast</b><br> | <div class="unterschrift"><b>Fig.2 Test digest of the POR level 2 targeted to the chloroplast</b><br> | ||
We digested this level 2 MoClo part with the restriction enzymes <i>Not</i>I and <i>Eco</i>RI.</div></p> | We digested this level 2 MoClo part with the restriction enzymes <i>Not</i>I and <i>Eco</i>RI.</div></p> | ||
| Line 43: | Line 43: | ||
<p>We tried to detected the expression the POR targeted to the chloroplast with HA-tag via immunoblotting.</p> | <p>We tried to detected the expression the POR targeted to the chloroplast with HA-tag via immunoblotting.</p> | ||
<p> | <p> | ||
| − | <img class="bild" src="https://static.igem.wiki/teams/4806/wiki/registry/level2/ | + | <img class="bild" src="https://static.igem.wiki/teams/4806/wiki/registry/level2/por-ctppsad-wb.png"> |
<div class="unterschrift"><b>Fig.3 Expression of the POR with the CTPPSAD-transit peptide</b><br> | <div class="unterschrift"><b>Fig.3 Expression of the POR with the CTPPSAD-transit peptide</b><br> | ||
(a)Level 2 MoClo construct for expression of the POR containing the CTPPSAD-tranist peptide with HA-tag was designed (see Fig.1 for part description) <br> (b) Picture of resulting western blot. The white arrow marks a cross reaction of antibodies. For reference, the UVM4 recipient strain and a strain expressing the HA-tagged ribosomal chloroplast 50S protein L5 (RPL5) were used as a negative and positive control, respectively | (a)Level 2 MoClo construct for expression of the POR containing the CTPPSAD-tranist peptide with HA-tag was designed (see Fig.1 for part description) <br> (b) Picture of resulting western blot. The white arrow marks a cross reaction of antibodies. For reference, the UVM4 recipient strain and a strain expressing the HA-tagged ribosomal chloroplast 50S protein L5 (RPL5) were used as a negative and positive control, respectively | ||
Revision as of 10:44, 19 September 2023
POR gene for expression in the chloroplast for Chlamydomonas reinhardtii (Phytobrick)
This composite part contains the PSAD-promotor (BBa_K4806010), the CTPPSAD-transit peptide (BBa_K4806014), the POR coding sequence (BBa_K4806003), the HA-tag (BBa_K3002017)* for detection and the tRPL23-terminator (BBa_K3002006)*. This part is codon-optimized for Chlamydomonas reinhardtii and was built as part of the CYPurify Collection. This level 2 part leads to potential detoxification of specific chemicals (Ohkawa & Inui, 2015).
Construct
This construct was designed using the modular cloning system (MoClo).
The resistance cassette for hygromycin is already built in the level 2 vector pMBS810 we are using. The usage of this vector allows the direct assembly of level 0 parts to level 2 constructs, facilitating the cloning time (Niemeyer & Schroda, 2022).
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 1535
Illegal PstI site found at 2795
Illegal PstI site found at 2855
Illegal PstI site found at 3514 - 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 2720
Illegal PstI site found at 1535
Illegal PstI site found at 2795
Illegal PstI site found at 2855
Illegal PstI site found at 3514
Illegal NotI site found at 3160 - 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 1535
Illegal PstI site found at 2795
Illegal PstI site found at 2855
Illegal PstI site found at 3514 - 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 1535
Illegal PstI site found at 2795
Illegal PstI site found at 2855
Illegal PstI site found at 3514
Illegal NgoMIV site found at 4443 - 1000COMPATIBLE WITH RFC[1000]
Results
We confirmed that this construct is built correctly via agarose gel electrophoresis.
We digested this level 2 MoClo part with the restriction enzymes NotI and EcoRI.
The test digest in Fig.2 was compared to an in-silico digest. Together with the sequencing results we were able to demonstrate that our construct was built correctly.
We tried to detected the expression the POR targeted to the chloroplast with HA-tag via immunoblotting.
(a)Level 2 MoClo construct for expression of the POR containing the CTPPSAD-tranist peptide with HA-tag was designed (see Fig.1 for part description)
(b) Picture of resulting western blot. The white arrow marks a cross reaction of antibodies. For reference, the UVM4 recipient strain and a strain expressing the HA-tagged ribosomal chloroplast 50S protein L5 (RPL5) were used as a negative and positive control, respectively
For detection the UVM4 strain was transformed with the construct in (a). 30 hygromycin-resistant transformants were cultivated in TAP medium and samples were taken after 3 days. Whole-cell proteins were extracted and analyzed by SDS-PAGE and immunoblotting using an anti-HA antibody. The expression of the POR (~ 77 kDa) is not visible.
Contribution
The * marked parts were not created by us. Our results can be found on the experience page of each part.