Difference between revisions of "Part:BBa K4197003"
Line 37: | Line 37: | ||
− | <h2> | + | <h2>Validation</h2> |
− | <p> | + | <p>Successful colonies were further tested by digestion with NotI which cut both in the plasmid and in the mRFP1 gene (Figure 2). Correct sizes were expected to be |
+ | 1178 and 6791 bp for Ara h 2.</p> | ||
<div class="center"> | <div class="center"> | ||
<div class="thumb tnone"> | <div class="thumb tnone"> | ||
<div class="thumbinner" style="width:80%;"> | <div class="thumbinner" style="width:80%;"> | ||
− | <a href=" | + | <a href="https://static.igem.wiki/teams/4197/wiki/parts/raph/anao3-dig.png" class="image"> |
− | <img alt="" src="https://static.igem. | + | <img alt="" src="https://static.igem.wiki/teams/4197/wiki/parts/raph/anao3-dig.png" width="100%" height=auto class="thumbimage" /></a> <div class="thumbcaption"> |
<div class="magnify"> | <div class="magnify"> | ||
− | <a href=" | + | <a href="https://static.igem.wiki/teams/4197/wiki/parts/raph/anao3-dig.png" class="internal" title="Enlarge"></a> |
</div> | </div> | ||
− | <b>Figure 2: </b> <b> | + | <b>Figure 2: </b> <b>Digestion by NotI of Ana o 3 with mRFP1 insertion.</b> |
− | + | The digestion product was checked with 0.6% agarose electrophoresis gel and revealed with EtBr. A theoretical gel is presented with each gel and the | |
+ | NEB 1 kb DNA ladder is used for the experimental gels (note that a different ladder is presented on the theoretical gel). colonies 21 and 23 present | ||
+ | the correct size for Ana o 3. | ||
</div> | </div> | ||
</div> | </div> | ||
</div> | </div> | ||
</div> | </div> | ||
+ | |||
+ | <p>This construction has not shown red fluorescence on the microscope. After sequencing, a mutation has been revealed on the mRFP sequence which does not allow us | ||
+ | to continue further with this construction.</p> | ||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
<h2>References</h2> | <h2>References</h2> | ||
<ol> | <ol> | ||
<i> | <i> | ||
− | + | <li> <partinfo>K4197012</partinfo> </li> | |
− | + | <li> <partinfo>K4197007</partinfo> </li> | |
− | + | <li> <a href="https://2022.igem.wiki/toulouse-insa-ups/index.html"> DAISY (INSA-UPS 2022)</a> </li> | |
− | </i> | + | </i> |
</ol> | </ol> | ||
+ | |||
+ | |||
</html> | </html> | ||
Revision as of 17:24, 8 October 2022
Ana o 3 expression at the surface of E. coli cells sortable by FACS using mRFP1
Brick expressing Ana o 3 at the surface of E. coli cell sortable by FACS
Introduction
The part expressing the gene of cashew nut Ana o 3 (
Construction
The ihfb800-mRFP1 construction was amplified by PCR with the high-fidelity Phusion polymerase using BFP/RFP IF+BglII R site R
(cgcgggatcgagatctatcacgaggcagaatttcagat) and BFP/RFP IF+BglII R site F (tagaggatcgagatctctgaaacagtgcaaagctaaccc) primers. The expected size of the amplicon is 1622
bp. The fragment was inserted on the linearized plasmid pET21b(+) with Ana o 3 (
The resulting products were transformed into Stellar cells and transformants were selected. Colonies were screened by PCR using screening_insert_R (ccgaaacaagcgctcatgagc) and screening_insert_F (ggttatgctagttattgctcagc) primers. The expected sizes of amplicons was 2944 bp with RFP and 1453 bp without RFP.
Validation
Successful colonies were further tested by digestion with NotI which cut both in the plasmid and in the mRFP1 gene (Figure 2). Correct sizes were expected to be 1178 and 6791 bp for Ara h 2.
This construction has not shown red fluorescence on the microscope. After sequencing, a mutation has been revealed on the mRFP sequence which does not allow us to continue further with this construction.
References
-
K4197012 -
K4197007 - DAISY (INSA-UPS 2022)
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 1527
Illegal EcoRI site found at 1741
Illegal XbaI site found at 1512
Illegal XbaI site found at 1658
Illegal PstI site found at 213
Illegal PstI site found at 224
Illegal PstI site found at 342 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 1527
Illegal EcoRI site found at 1741
Illegal NheI site found at 1703
Illegal PstI site found at 213
Illegal PstI site found at 224
Illegal PstI site found at 342
Illegal NotI site found at 1519
Illegal NotI site found at 2697 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 1527
Illegal EcoRI site found at 1741
Illegal BglII site found at 1592
Illegal BamHI site found at 1735
Illegal XhoI site found at 2706 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 1527
Illegal EcoRI site found at 1741
Illegal XbaI site found at 1512
Illegal XbaI site found at 1658
Illegal PstI site found at 213
Illegal PstI site found at 224
Illegal PstI site found at 342 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 1527
Illegal EcoRI site found at 1741
Illegal XbaI site found at 1512
Illegal XbaI site found at 1658
Illegal PstI site found at 213
Illegal PstI site found at 224
Illegal PstI site found at 342
Illegal AgeI site found at 329
Illegal AgeI site found at 1360
Illegal AgeI site found at 1472 - 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 2368