Difference between revisions of "Part:BBa K3002001:Experience"
 
(One intermediate revision by one other user not shown)
Line 6: Line 6:
 
===Applications of BBa_K3002001===
 
===Applications of BBa_K3002001===
 
<html>
 
<html>
 +
<h1>Cytosolic Expression
 +
                </h1>
 
<p></p><div class="figure">
 
<p></p><div class="figure">
−
<img src="https://2019.igem.org/wiki/images/4/42/T--TU_Kaiserslautern--resultsFigure2.svg"/>
+
<img src="https://2019.igem.org/wiki/images/1/1b/T--TU_Kaiserslautern--resultsFigure4.svg"/>
−
<p class="caption"><span class="phat">Toxicity test with TPA, EG and EtOH.           
+
<p class="caption"><span class="phat">Expression of the enzymes MUT-PETase and MHETase in <i>Chlamydomonas</i> <i>reinhardtii</i>.            
−
</span>Growth of <i>Chlamydomonas</i> strain UVM4 was tested in TAP medium containing 0 mM (control), 5 mM and 10 mM terephtalic acid (TPA), ethylene glycol (EG) and ethanol (EtOH). The pH in the medium containing TPA was adjusted to pH 7 with potassium acetate. <span class="accent">(a)</span> UVM4 cells were inoculated in 20 mL with 2*105 cells/mL. Growth was measured by counting cells for 5 days. Error bars represent standard error of three biological replicates. <span class="accent">(b)</span> Spot test analysis with the same concentrations of EG, EtOH and TPA as used in <span class="accent">(a)</span>. Note that the UVM4 strain lacks a cell wall and that cells die if they are too diluted.
+
</span><span class="accent">(a)</span> Level 2 MoClo construct harboring the aadA selection marker and the coding sequences for MUT-PETase, and MHETase (see Figure 1 for part description). <span class="accent">(b)</span> The UVM4 strain was transformed with the construct shown in <span class="accent">(a)</span>. 11 spectinomycin-resistant transformants were inoculated in TAP and samples taken after 3 days. Extracted whole-cell proteins were analysed by SDS-PAGE and immunoblotting using an anti-HA antibody. MW – molecular weight. The black arrow represents the MHETase, the white arrow the MUT-PETase. The expression of both MHETase (~70 kDa) and MUT-PETase (~35 kDa) is visible in colonies 18, 22 and 27 (<a href="https://parts.igem.org/Part:BBa_K3002200">BBa_K3002200</a>). The UVM4 recipient strain and a strain expressing the HA-tagged ribosomal chloroplastic 50S protein L5 (RPL5) served as negative and positive controls, respectively.
 +
</p>
 +
</div><p>
 +
Both MUT-PETase and MHETase show cytosolic expression and secretion when containing the cCA secretion signal. The PSAD promoter is a reliable regulatory element for the aadA coding sequence and thus allows a selection of transformants.  
 
</p>
 
</p>
 
</div>
 
</div>

Latest revision as of 19:49, 13 December 2019


This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

Applications of BBa_K3002001

Cytosolic Expression

Expression of the enzymes MUT-PETase and MHETase in Chlamydomonas reinhardtii. (a) Level 2 MoClo construct harboring the aadA selection marker and the coding sequences for MUT-PETase, and MHETase (see Figure 1 for part description). (b) The UVM4 strain was transformed with the construct shown in (a). 11 spectinomycin-resistant transformants were inoculated in TAP and samples taken after 3 days. Extracted whole-cell proteins were analysed by SDS-PAGE and immunoblotting using an anti-HA antibody. MW – molecular weight. The black arrow represents the MHETase, the white arrow the MUT-PETase. The expression of both MHETase (~70 kDa) and MUT-PETase (~35 kDa) is visible in colonies 18, 22 and 27 (BBa_K3002200). The UVM4 recipient strain and a strain expressing the HA-tagged ribosomal chloroplastic 50S protein L5 (RPL5) served as negative and positive controls, respectively.

Both MUT-PETase and MHETase show cytosolic expression and secretion when containing the cCA secretion signal. The PSAD promoter is a reliable regulatory element for the aadA coding sequence and thus allows a selection of transformants.

User Reviews

UNIQdc3767a69421d9a6-partinfo-00000001-QINU UNIQdc3767a69421d9a6-partinfo-00000002-QINU