Part:BBa_K3278007
pDawn-NAS
The light inducible NAS plasmid: pDawn-NAS
We intended to use light to induce NAS expression instead of IPTG so that we can easily program light to control the production of N-palmitoyl serinol. In order to replace the lac operon with a light inducible promoter, we introduced the blue-light inducible system called pDawn into our system and constructed the pDawn-NAS fusion plasmid.
For cloning, we used PCR with high-fidelity enzyme to get the linear pDawn vector (7198 bp) and NAS gene fragment (1685bp) with overlapping sequence. As shown above, the PCR product was separated by agarose gel electrophoresis, then gel extraction kit was used to isolate the target DNA fragments. After that, Gibson Assembly Cloning kit was used to recombine the vector and fragment into the final plasmid pDawn-NAS. The recombination product was then transformed into DH5α competent cell. Finally, we got the our desired pDawn-NAS plasmid. The sequence map was shown below. The recombinant plasmid was then transformed into BL21:DE3 bacteria for gene expression.
The production of N-palmitoyl serinol by pDawn-NAS
As we have got our desired plasmid, we wanted to test whether NAS gene can be expressed to produce N-palmitoyl serinol in the pDawn system. Therefore, we directly used the LC-MS to determine the production of N-palmitoyl serinol. We inoculated 100 ul overnight cultured bacteria fluid into 10 ml LB with kanamycin (50 ug/ml) and cultured at 37 °C with shaking (220rpm) for 3 h until OD550 reaches 0.6-1.0. Then we poured all the LB into a petri dish and exposed the petri dish with blue light at 29 °C for 24 h. Next, we centrifuged down 1 ml bacteria to get the supernatant and added equal volume ethyl acetate to it. The ethyl acetate solution was finally used for the LC-MS. As shown blow, introduction of NAS in the pDawn successfully induced a strong signal around t = 4.12 min. Yet, the empty plasmid control showed little signal there.
We confirmed the t = 4.12 min peak is the N-palmitoyl serinol as the following. We fragmented the N-palmitoyl serinol by giving a certain amount of energy, and then detected the Mass/Charge value of each fragment. The results of standard N-palmitoyl serinol and the sample of small molecule at t = 4.12 min were shown blow. They had nearly the same characteristic patterns in peak positions and ratio of intensity, which proved that the signal at t = 4.12 min was generated by N-palmitoyl serinol. Thus, we successfully confirmed that the production of the desired molecule via the pDawn-NAS.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 3486
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 2148
Illegal BglII site found at 5098
Illegal BamHI site found at 3517
Illegal XhoI site found at 5172 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 46
Illegal NgoMIV site found at 178
Illegal NgoMIV site found at 272
Illegal NgoMIV site found at 565
Illegal NgoMIV site found at 1059
Illegal NgoMIV site found at 1077
Illegal NgoMIV site found at 1167
Illegal AgeI site found at 397
Illegal AgeI site found at 1525
Illegal AgeI site found at 4533
Illegal AgeI site found at 4659 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 1620
Illegal BsaI.rc site found at 508
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