Team:ShanghaiTech China/NAS
NAS gene in E. coli
N-acyl synthase (NAS) genes are found in intestinal bacteria. It can synthesize the N-acyl amine by a two-step reaction shown below.
Then, N-acyl amide interacts with G-Protein Coupled Receptor (GPCR) expressed in the intestine. When activated by N-acyl amide secreted by resident gut bacteria, it will help reduce the glucose level and contributes to the treatment of diabetes. There are many families of N-acyl amides, each of them may interact with different GPCRs. According to the experiment results, GPR119 activated by N-palmitoyl serinol shows the relevantly strongest interaction [1]. As GPR199 belongs to the GPCR family and plays an important role in regulating the glucose homeostasis, N-palmitoyl serinol was then chosen to be our desired small molecule agonist. We found the related NAS gene from GeneBank and synthesized the CDS and cloned it into the pET28c plasmid. Then, we transformed it into E. coli to acquire the engineering bacteria.
The expression of NAS gene induced by IPTG
In the pET28c-NAS plasmid, NAS expression can be induced by IPTG. To check the expression level of NAS gene (protein size ~62.086kDa) in E. coli, we transformed pET28c-NAS plasmid into E. coli BL21:DE3 which contains the T7 polymerase gene. The bacteria were then cultured in LB with kanamycin. On the next day, we inoculate 300 ul bacteria into 15 ml new LB broth, and cultured for 3 h. After that, we extracted 1.5 ml from 15 ml LB as 0 h sample. Then, we divided the remaining bacteria into two shaking tubes. Each tube contained 6-ml LB. IPTG (1mM) was added into one of the tubes. Then, we extracted 1.5 ml at a certain time (3 h, 7 h, 22 h post IPTG induction) and centrifuged down the bacteria. Next, we ran SDS PAGE to determine the NAS expression level. For SDS-PAGE, we resuspended the bacteria with 100 ul PBS. We then added 25ul 5 X SDS loading buffer and boiled the samples at 100 °C for 15 min. After that, we centrifuged at 13000 rpm, 10 min to get rid of the debris, and loaded liquid supernatant for SDS PAGE. The SDS PAGE result is shown below.
The production of N-palmitoyl serinol
As we successfully confirmed the expression of NAS gene, we need to know whether N-palmitoyl serinol can be synthesized and secreted out of the E. coli. Therefore, we used liquid chromatograph-mass spectrometer (LC-MS) to determine the existence of N-palmitoyl serinol. We extracted 1 ml overnight cultured bacteria fluid into 50 ml LB with kanamycin (50ug/ml) and IPTG (25 uM) and further cultured for 4 days at 30 °C with shaking (200rpm). After that, we extracted 1 ml bacteria and centrifuged down the bacteria to get the supernatant. Then, we added equal volume ethyl acetate to extract the N-palmitoyl serinol from LB supernatant. The Ethyl acetate solution was used to run the LC-MS.
As for LC-MS, structurally different small molecules will be detected at different reaction times. Therefore, signals at different reaction time on the abscissa represent one type of small molecule. First, we ran a standard N-palmitoyl serinol as a positive control. The LC-MS result of the standard N-palmitoyl serinol (approximately 0.05 ug/ul, molecular weight 330.3 g/mol) is shown below. The result indicates that N-palmitoyl serinol signal was about 4.30 min.
After testing the standard solution, we ran the samples of bacteria supernatant from pET28c-NAS and empty pET28c vector by LC-MS. The results showed that the pET28c-NAS group generated a strong signal at the response time of 4.31 min. While the empty control group had nearly no signal at around 4.30 min. Therefore, we obtained the desired N-palmitoyl serinol product secreted by our engineered bacteria.
References
[1] Cohen, L. J. et al. Commensal bacteria make GPCR ligands that mimic human signalling molecules. Nature 549, 48-+, doi:10.1038/nature23874 (2017).