Difference between revisions of "Part:BBa K4806000"
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<p>10 hplc erythromycin xxxxxxxxxxxxxxxxxxxxxxxxxx </p>
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<p>We tried to detect the activity of CYP3A4 with HA-tag (<a href="https://parts.igem.org/Part:BBa_K4806200">BBa_K4806200</a>) via primary antibody.</p>
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  <img class="bild" src="https://static.igem.wiki/teams/4806/wiki/website/resu/ery.png">
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  <div class="unterschrift"><b>Fig.12 HPLC Erythromycin</b><br>
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<p>After testing several antibody concentrations, we can conclude that the commercial CYP3A4 antibody is not capable of detecting CYP3A4 produced in Chlamydomonas. </p>
  
 
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<p>We tried to detect the activity of CYP3A4 with HA-tag (<a href="https://parts.igem.org/Part:BBa_K4806200">BBa_K4806200</a>) via primary antibody.</p>
 
<p>We tried to detect the activity of CYP3A4 with HA-tag (<a href="https://parts.igem.org/Part:BBa_K4806200">BBa_K4806200</a>) via primary antibody.</p>
 
<p>
 
<p>
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   <img class="bild" src="https://static.igem.wiki/teams/4806/wiki/website/resu/fig-16.jpgg">
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   <img class="bild" src="https://static.igem.wiki/teams/4806/wiki/website/resu/fig-16.jpg">
 
   <div class="unterschrift"><b>Fig.12 CYP3A4 antibody test</b><br>
 
   <div class="unterschrift"><b>Fig.12 CYP3A4 antibody test</b><br>
 
   (a) Level 2 MoClo constructs for the production 3xHA-tagged CYP3A4. Whole-cell proteins of transformants E2, E13, and E26 producing 3xHA-tagged CYP3A4 were extracted and analyzed by SDS-PAGE and immunoblotting using a commercial CYP3A4 antibody at different dilutions: (b) 1:1000, (c) 1:5000, (d) 1:10000, (e) 1:20000. The black arrow marks CYP3A4 and the white arrow marks a cross-reaction of the antibody. UVM4 served as negative control. (f) To confirm the expression of HA-tagged CYP3A4, the blot was detected with an anti-HA antibody.</div>
 
   (a) Level 2 MoClo constructs for the production 3xHA-tagged CYP3A4. Whole-cell proteins of transformants E2, E13, and E26 producing 3xHA-tagged CYP3A4 were extracted and analyzed by SDS-PAGE and immunoblotting using a commercial CYP3A4 antibody at different dilutions: (b) 1:1000, (c) 1:5000, (d) 1:10000, (e) 1:20000. The black arrow marks CYP3A4 and the white arrow marks a cross-reaction of the antibody. UVM4 served as negative control. (f) To confirm the expression of HA-tagged CYP3A4, the blot was detected with an anti-HA antibody.</div>

Revision as of 14:55, 12 October 2023


CYP3A4 gene for Chlamydomonas reinhardtii (Phytobrick)

This basic part contains the coding sequence of CYP3A4 (B3-B4). This part is codon-optimized for Chlamydomonas reinhardtii and was built as part of the CYPurify Collection. In combination with a promoter like AβSAP(i) (BBa_K4806013) and a terminator like tRPL23 (BBa_K3002006)*, this level 0 part leads to expression and potential detoxification of specific chemicals (Ohkawa & Inui, 2015). To detect the target protein a tag like HA-tag (BBa_K3002017)* is recommended.


Constructs

Fig.1 Construct design
We designed 6 level 2 constructs containing CYP3A4 using the modular cloning system (MoClo).


Here are the links to the built constructs:

  • 1. CYP3A4 gene with FLAG-tag for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806201)
  • 2. CYP3A4 gene with mStop for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806202)
  • 3. CYP3A4 gene with HA-tag for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806200)
  • 4. CYP3A4 gene with mNeonGreen for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806204)
  • 5. CYP3A4 gene for expression in the chloroplast for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806203)
  • 6. CYP3A4 tandem for expression together with the POR for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806214)

These constructs were transformed into Chlamydomonas reinhardtii. Besides the CYP3A4 coding sequence the constructs contain either the AβSAP(i)-promotor (BBa_K4806013) or the PSAD-promotor (BBa_K4806010),either the FLAG-tag (BBa_K4806012), the HA-tag (BBa_K3002017)* or mNeonGreen (BBa_K4806006) for detection or mStop (BBa_K4806009) and the tRPL23-terminator (BBa_K3002006)*. Additionally, one construct contains the CTPPSAD transit peptide to the chloroplast (BBa_K4806014). The resistance cassette for spectinomycin is already built in the level 2 vector pMBS807 we are using (exept for the tandem construct). 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


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 1104
    Illegal PstI site found at 1426
    Illegal PstI site found at 1486
    Illegal PstI site found at 1958
    Illegal PstI site found at 2027
    Illegal PstI site found at 2131
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 1104
    Illegal PstI site found at 1426
    Illegal PstI site found at 1486
    Illegal PstI site found at 1958
    Illegal PstI site found at 2027
    Illegal PstI site found at 2131
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 1104
    Illegal PstI site found at 1426
    Illegal PstI site found at 1486
    Illegal PstI site found at 1958
    Illegal PstI site found at 2027
    Illegal PstI site found at 2131
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 1104
    Illegal PstI site found at 1426
    Illegal PstI site found at 1486
    Illegal PstI site found at 1958
    Illegal PstI site found at 2027
    Illegal PstI site found at 2131
    Illegal NgoMIV site found at 1348
  • 1000
    COMPATIBLE WITH RFC[1000]


Results

We detected the expression of CYP3A4 with HA-tag (BBa_K4806200) via immunoblotting.

Fig.2 Expression of CYP3A4 with HA-tag
(a)Level 2 MoClo construct for expression of the enzyme CYP3A4 containing the HA-tag was designed (see Fig.1 for part description)
(b) Picture of resulting western blot. The enzyme CYP3A4 is marked by a black arrow, 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 spectinomycin-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 CYP3A4 (~ 57 kDa) is visible.


We detected the expression of CYP3A4 with FLAG-tag (BBa_K4806201) via immunoblotting.

Fig.3 Expression of CYP3A4 with FLAG-tag
(a)Level 2 MoClo construct for expression of the enzyme CYP3A4 containing the FLAG-tag was designed (see Fig.1 for part description)
(b) Picture of resulting western blot. The enzyme CYP3A4 is marked by a black arrow, the white arrow marks a cross reaction of antibodies. For reference, the UVM4 recipient strain and a strain expressing the FLAG-tagged VIPP1 were used as a negative and positive control, respectively.

For detection the UVM4 strain was transformed with the construct in (a). 30 spectinomycin-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-FLAG antibody. The expression of CYP3A4 (~ 57 kDa) is visible.


We detected the expression of CYP3A4 tandem together with the POR with HA-tag (BBa_K4806214) via immunoblotting.

Fig.4 Expression of CYP3A4 tandem together with the POR with HA-tag
(a)Level 2 MoClo construct for expression of the enzyme CYP3A4 tandem together with the POR containing the HA-tag was designed (see Fig.1 for part description)
(b) Picture of resulting western blot. The enzyme CYP3A4/POR is marked by a black arrow, 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 CYP3A4 (~ 57 kDa) and the POR (~77 kDa) is visible.


We tried to supertransform the POR with HA-tag (BBa_K4806209) into positive CYP3A4 strains.

Fig.5 Supertrafo of the POR into positive CYP3A4 strains
(a) Level 2 MoClo constructs for expression of the enzymes CYP3A4 and the POR containing the HA-tag. (b) The CYP3A4 strain was transformed with the POR construct in (a). 30 hygromomycin-resistant transformants were cultivated in TAP-medium and samples taken after 3 days. Whole-cell proteins were extracted and analyzed by SDS-PAGE and immunoblotting using an anti-HA antibody. In the resultant the white arrow marks a cross reaction of antibodies. The expression of CYP3A4 (~57 kDa) is visible. The expression of the POR (~ 77 kDa) is not visible. For reference, the UVM4 recipient strain and a strain expressing the HA-tagged ribosomal chloroplastic 50S protein L5 (RPL5) were used as a negative and positive control, respectively.

Sadly we were not able to detect the expression of the POR.


We detected that the construct CYP3A4 with HA-tag (BBa_K4806200) is correctly embedded within the membrane via freeze-thaw assay and immunoblotting.

Fig.6 Freeze thaw assay of CYP3A4 with HA-tag
(a) Level 2 MoClo construct for expression of the enzyme CYP3A4 containing the HA-tag was designed. (b) The UVM4 strain was transformed with the construct in (a). An anti-HA antibody was used to detect the enzyme CYP3A4 (A). For control an anti-Cytf antibody was used to detect the membrane bound proteins and a CGE1-antibody for soluble proteins (B). 30 spectinomycin-resistant transformants were cultivated in TAP-medium and samples taken after. 3 days. For reference whole-cell proteins were extracted and analyzed by SDS-PAGE and immunoblotting. The other samples were treated according to protocol and analyzed also by SDS-PAGE and immunoblotting. The expression of CYP3A4 (~57 kDa) is visible in the pellet (P) and not in the supernatant (S) confirming the membrane intercalation. For reference, the UVM4 strain was used as a negative control.

We were able to detect the expression of CYP3A4 within the pellet, demonstrating that our CYP is correctly embedded within the membrane.


We tried to detect the activity of CYP3A4 with HA-tag (BBa_K4806200) by incubating them in the antibiotic Erythromycin.

Fig.7 Growth assay
(a) Level 2 MoClo construct for expression of the enzyme CYP3A4 containing the HA-tag. (b) Positive CYP3A4 transformants.

Our initial assumption was that this test yielded a positive result due to the increased slope of the E26 strain and the associated faster grwoth. Unfortunately, this strain also grows faster without erythromycin, which does not provide a conclusive indication of activity. The E2 strain even exhibits greater sensitivity to erythromycin compared to the wildtype UVM4.


We tried to detect the activity of CYP3A4 with HA-tag (BBa_K4806200) by incubating UVM4 strains in medium previously incubated by our transformants.

Fig.8 Survivability assay
(a) Level 2 MoClo construct for expression of the enzyme CYP3A4 containing the HA-tag. (b) Positive CYP3A4 transformants were treated with 10 mg/l erythromycin at a high cell density and incubated for 48 hours. The cells were subsequently centrifuged and removed. (c) The supernatant from each culture was combined with UVM4. The figure illustrates the status after 96 h of incubation with the UVM4 strain. (d) After an incubation period of 336 hours, the Chlamys have regrown and reached a stationary state.

This approach also proved unsuccessful, as all cultures, including the negative control, developed resistance to erythromycin after approximately 1.5 weeks of incubation.


We tried to detect the activity of CYP3A4 with HA-tag (BBa_K4806200) via drop-test.

Fig.9 Drop-test
(a) Level 2 MoClo construct for expression of the enzyme CYP3A4 containing the HA-tag. (b) 10 µl positive CYP3A4 transformants were dropped on TAP-plates as control. (c) 10 µl positive CYP3A4 transformants were dropped on TAP-plates containing 8 mg/l erythromycin. UVM4 strains were used as negative control.

We dropped all our positive CYP3A4 transformants with HA-tag on plates with and without erythromycin. At this erytrhomycin concentration no wildtype should grow, as previously tested. As the incubation time of this test was much longer than the testing, spontaneous mutations occured in the nagtive control. Therefore this experiment does not verify activity of our enzymes.


We tried to detect the activity of CYP3A4 with HA-tag (BBa_K4806200) via primary antibody.

Fig.12 HPLC Erythromycin
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After testing several antibody concentrations, we can conclude that the commercial CYP3A4 antibody is not capable of detecting CYP3A4 produced in Chlamydomonas.


We tried to detect the activity of CYP3A4 with mStop (BBa_K4806202) via drop-test.

Fig.11 Drop-test
(a) Level 2 MoClo construct for expression of the enzyme CYP3A4 containing mStop. (b) 10 µl CYP3A4 transformants were dropped on TAP-plates containing 8 mg/l erythromycin. UVM4 strains were used as negative control.

We dropped all our CYP3A4 transformants with mStop on plates with and without erythromycin. At this erytrhomycin concentration no wildtype should grow, as previously tested. As the incubation time of this test was much longer than the testing, spontaneous mutations occured in the nagtive control. Therefore this experiment does not verify activity of our enzymes.


We tried to detect the activity of CYP3A4 with HA-tag (BBa_K4806200) via primary antibody.

Fig.12 CYP3A4 antibody test
(a) Level 2 MoClo constructs for the production 3xHA-tagged CYP3A4. Whole-cell proteins of transformants E2, E13, and E26 producing 3xHA-tagged CYP3A4 were extracted and analyzed by SDS-PAGE and immunoblotting using a commercial CYP3A4 antibody at different dilutions: (b) 1:1000, (c) 1:5000, (d) 1:10000, (e) 1:20000. The black arrow marks CYP3A4 and the white arrow marks a cross-reaction of the antibody. UVM4 served as negative control. (f) To confirm the expression of HA-tagged CYP3A4, the blot was detected with an anti-HA antibody.

After testing several antibody concentrations, we can conclude that the commercial CYP3A4 antibody is not capable of detecting CYP3A4 produced in Chlamydomonas.


We tried to detect the activity of CYP3A4 with HA-tag (BBa_K4806200) via HPLC.

Fig.13 Estradiol HPLC
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We tried to detect the activity of CYP3A4 with HA-tag (BBa_K4806200) via absorption measurements

Fig.14 Absorption measurements
CYP3A4 expressing lines E7 and E26 with HA-tag were prepared in a non-degenerating way, to ensure correct protein structure. Their absorption was then measured at the Nano Drop 2000. As negative control served UVM4.

Figure 14 demonstrates that the peak at 418 nm, normalized to the total protein content, is elevated in the transformants compared to the wild type, indicating that this new method is valid for screening untagged constructs, but there still a lot more tests to do to verify this method.


Contribution

The * marked parts were not created by us. Our results can be found on the experience page of each part.


The CYurify Collection

The world is at a crossroad. We must decide now how we want to continue living in order to survive. To contribute to this cause, we proudly present our CYPURIFY Collection for Chlamydomonas reinhardtii. The contamination of our water with toxic substances is on the rise, damaging ecosystems and eventually impacting us humans. We see it as our duty to take action.

To accomplish this, we designed 23 level 0, 9 level 1 and 24 level 2 parts for bioremediation of toxic wastewater using Modular Cloning. At heart of this collection are the Cytochrome P450 enzymes. Some of these monooxygenases are already used in synthesis or in medicine. We aimed to take a further step in research by expressing these enzymes in Chlamydomonas for the first time.

Chlamydomonas reinhardtii is the perfect fit for our system as a phototrophic organism with cost-effective and sustainable cultivation. Additionally, this organism is well-studied and easy to transform. We have access to a vast library of preexisting parts, all compatible with Modular Cloning.

Modular Cloning is a cloning method based on the Golden Gate System. What makes it unique is the ability to assemble entire genes in a single reaction. This is made possible by using type IIS restriction enzymes, which cut outside their recognition sequence, effectively removing it after ligation into the target vector. Therefore, the reaction proceeds in a specific direction. The parts are divided into level 0,1 and 2. Level 0 parts are basic components such as promotors, terminators or tags. Level 1 parts are combinations of these level 0 parts, forming transcriptional units. Level 2 parts are combinations of level 1 parts, allowing the expression of multiple genes simultaneously. Level 0 parts are assigned one of 10 positions, with standardized overhangs between them, enabling the exchange of parts between laboratories.

With our collection, we aim to contribute to environmental protection. This collection is infinitely expandable with new CYPs that can degrade other toxic substances. So, what are you waiting for?