Difference between revisions of "Part:BBa K3166004"
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<p>pETDuet-1T7-thSQS-2T7-CrtN(pET-thN) BBa_K3166998</p> | <p>pETDuet-1T7-thSQS-2T7-CrtN(pET-thN) BBa_K3166998</p> | ||
<p>pETDuet-rbs-idi-rbs-ispA-rbs-YSS-2T7-CrtN（best composite part BBa_K3166808https://parts.igem.org/Part:BBa_K3166058）</p> | <p>pETDuet-rbs-idi-rbs-ispA-rbs-YSS-2T7-CrtN（best composite part BBa_K3166808https://parts.igem.org/Part:BBa_K3166058）</p> | ||
| − | + | <p>1. Target genes YSS, NSS, KSS, thSQS and crtN were obtained;</p> | |
| − | + | <p>High-fidelity DNA polymerase was used to conduct PCR on genes YSS, NSS, KSS, thSQS and crtN. PCR results were as follows. The gel of PCR products was recycled to obtain DNA.</p> | |
| − | + | ||
https://2019.igem.org/wiki/images/7/78/T--SEFLS_Shanghai--BBa_all-fig4.jpg | https://2019.igem.org/wiki/images/7/78/T--SEFLS_Shanghai--BBa_all-fig4.jpg | ||
| + | <p>2. Double enzyme digestion of plasmid pETDuet-1 at MCS1 site was performed, and skeleton vector was obtained after DNA gel recycling. The following figure shows plasmid vector and gene fragment after gel recycling.</p> | ||
https://2019.igem.org/wiki/images/1/17/T--SEFLS_Shanghai--BBa_all-fig5.jpg | https://2019.igem.org/wiki/images/1/17/T--SEFLS_Shanghai--BBa_all-fig5.jpg | ||
| + | <p>3. Gibson assembly kit was used to connect the target fragments YSS, NSS, KSS and thSQS into the MCS1 site of petduet-1, respectively. The PCR results of bacterial solution were as follows:</p> | ||
https://2019.igem.org/wiki/images/0/03/T--SEFLS_Shanghai--BBa_all-fig6.jpg | https://2019.igem.org/wiki/images/0/03/T--SEFLS_Shanghai--BBa_all-fig6.jpg | ||
| + | <p>4. Extract the plasmid of the verified positive transformants to prepare the connection of crtN gene next;</p> | ||
| + | <p>5. The plasmid extracted from positive transformants was digested with double enzyme digestion at MCS2 site and connected with crtN gene after the gel recycling. The following figure shows the plasmid fragment and gene crtN after enzyme digestion.</p> | ||
https://2019.igem.org/wiki/images/f/f5/T--SEFLS_Shanghai--BBa_all-fig7.jpg | https://2019.igem.org/wiki/images/f/f5/T--SEFLS_Shanghai--BBa_all-fig7.jpg | ||
| + | <p>6. The monoclone grown after the connection and transformation was verified by PCR, and the results were as follows: the positive transformant was simultaneously connected to the target gene and crtN gene;</p> | ||
https://2019.igem.org/wiki/images/0/0a/T--SEFLS_Shanghai--BBa_all-fig8.jpg | https://2019.igem.org/wiki/images/0/0a/T--SEFLS_Shanghai--BBa_all-fig8.jpg | ||
| + | <p>7. The plasmids pET-KN, pET-NN, pET-YN and pET-thN of the positive transformant were extracted. The extracted plasmid and p35151 were co-transformed into BL21 (DE3) competent cell.</p> | ||
| + | <p>8. Monoclone was selected for fermentation in TB medium, and cultured at 180 r/min, 30°C for 48h after the induction by adding 0.1mm IPTG.</p> | ||
| + | <p>9. Bacteria were collected 48h later for carotenoid extraction and detection.</p> | ||
| + | <p>10. The results showed that the activity of squalene synthase YSS was the highest.</p> | ||
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Revision as of 02:47, 22 October 2019
dehydrosqualene desaturase CrtN
Dehydrosqualene desaturase CrtN helps transform squalene into diaponeurosporene.
Usage and Biology
It was reported that the dehydrosqualene desaturase CrtN from Staphylococcus aureus converts squalene into yellow carotenoid pigments, permitting the colorimetric detection of squalene produced in the cell. Sub-cloning CrtN into the plasmid helps us to screen out squalene synthase with high activity rapidly.
It was sub-cloned into genes below:
pETDuet-1T7-KSS-2T7-CrtN(pET-KN) BBa_K3166997
pETDuet-1T7-NSS-2T7-CrtN(pET-NN) BBa_K3166800
pETDuet-1T7-YSS-2T7-CrtN(pET-YN) BBa_K3166998
pETDuet-1T7-thSQS-2T7-CrtN(pET-thN) BBa_K3166998
pETDuet-rbs-idi-rbs-ispA-rbs-YSS-2T7-CrtN（best composite part BBa_K3166808https://parts.igem.org/Part:BBa_K3166058）
1. Target genes YSS, NSS, KSS, thSQS and crtN were obtained;
High-fidelity DNA polymerase was used to conduct PCR on genes YSS, NSS, KSS, thSQS and crtN. PCR results were as follows. The gel of PCR products was recycled to obtain DNA.
2. Double enzyme digestion of plasmid pETDuet-1 at MCS1 site was performed, and skeleton vector was obtained after DNA gel recycling. The following figure shows plasmid vector and gene fragment after gel recycling.
3. Gibson assembly kit was used to connect the target fragments YSS, NSS, KSS and thSQS into the MCS1 site of petduet-1, respectively. The PCR results of bacterial solution were as follows:
4. Extract the plasmid of the verified positive transformants to prepare the connection of crtN gene next;
5. The plasmid extracted from positive transformants was digested with double enzyme digestion at MCS2 site and connected with crtN gene after the gel recycling. The following figure shows the plasmid fragment and gene crtN after enzyme digestion.
6. The monoclone grown after the connection and transformation was verified by PCR, and the results were as follows: the positive transformant was simultaneously connected to the target gene and crtN gene;
7. The plasmids pET-KN, pET-NN, pET-YN and pET-thN of the positive transformant were extracted. The extracted plasmid and p35151 were co-transformed into BL21 (DE3) competent cell.
8. Monoclone was selected for fermentation in TB medium, and cultured at 180 r/min, 30°C for 48h after the induction by adding 0.1mm IPTG.
9. Bacteria were collected 48h later for carotenoid extraction and detection.
10. The results showed that the activity of squalene synthase YSS was the highest.