Part:BBa_K5300039
PnifH-dipeptide aldehyde, composite part composed of PnifH and dipeptide aldehyde biosynthetic gene
We constructed a composite circuit combining the PnifH and dipeptide aldehyde biosynthetic gene cluster. This circuit was introduced into Sinorhizobium fredii CCBAU45436 in the legume-nodule symbiosis system for plant-based validation. Initially, we introduced only half of the dipeptide aldehyde synthesis gene cluster, which cannot finish the synthesis of dipeptide aldehyde. This was done to test whether the plasmid insertion would exert growth pressure on the nodules. Afterwards, the complete dipeptide aldehyde synthesis gene cluster was introduced into our chassis to further verify whether the chassis containing recombinant plasmid could successfully produce the target product.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NotI site found at 8611
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 2543
Illegal BglII site found at 3707
Illegal BglII site found at 4379
Illegal BglII site found at 6047 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 2965
Illegal NgoMIV site found at 3327
Illegal NgoMIV site found at 4498
Illegal NgoMIV site found at 5275
Illegal NgoMIV site found at 6058
Illegal NgoMIV site found at 8262
Illegal NgoMIV site found at 8295 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 2016
Illegal BsaI site found at 2481
Illegal BsaI site found at 3573
Illegal BsaI site found at 6726
Illegal BsaI site found at 7755
Illegal BsaI.rc site found at 4771
Illegal BsaI.rc site found at 6394
Illegal BsaI.rc site found at 6511
Illegal SapI site found at 4035
Illegal SapI.rc site found at 1959
Plasmid construction and validation
In order to verify that our idea works properly, we first conducted the synthesis of dipeptide aldehydes. We constructed a composite circuit of nifH promoter and dipeptide aldehyde synthesis gene cluster (Figure 3-1-1). To verify whether the plasmid would stress the growth of nodules, we constructed a composite circuit of the nifH promoter and half of the sequence of the dipeptidyl aldehyde synthesis gene cluster (Fig. 3-1-2), which encodes a product that cannot synthesize complete dipeptidyl aldehyde.
The plasmid for dipeptide aldehyde synthesis in E. coli was transferred into Sinorhizobium fredii CCBAU45436 using triparental mating. The verified correct strain was inoculated into Jidou 17 and harvested after 21 days of growth, and photographs were taken to record the above-ground phenotype, below-ground phenotype (Fig. 3-1-3), and nodules section (Fig. 3-1-4).
For phenotypes and nodule sections, there were no significant differences between plants inoculated with wild type and strains containing dipeptide aldehyde synthesis plasmid. Their chlorophyll content in leaves and dry weight of above-ground parts were examined (Figure 3-1-5).
For chlorophyll content of plants after 21 days of growth, there was a significant difference between plants inoculated with rhizobium and the blank control. There was no significant difference between the different strains. For dry weight of above-ground portions, inoculation with recombinant-plasmid-containing bacteria led to a decrease in dry weight, whereas whether or not dipeptidyl aldehydes were synthesized did not affect dry weight of above-ground portions.
Real-time fluorescence quantitative PCR
We used RT-qPCR to quantify gene transcription levels. We extracted RNA from +bgc33-sfp strain and wild-type strain grown for 21 days respectively and conducted reverse transcription. qPCR was performed using cDNA as a template. 16S rRNA was used as an internal reference to compare the expression of the target gene in the +bgc33-sfp strain with the endogenous nifH gene. We designed three pairs of primers (bgc33-a, bgc33-b, and bgc33-c) for bgc33 gene to characterize the transcript levels of different regions of the gene in terms of the transcript levels of three fragments, and performed three biological replicates (Figure 3-2-1).
Obviously, we found that the expression of bgc33 gene decreased following the increase of its distance from the promoter. The transcript levels of the first two segments of bgc33 gene were up-regulated compared to the expression of the endogenous nifH gene, while the expression of the third segment was down-regulated. We hypothesized that the decreased transcript levels of the second half of the gene may be due to the fact that the gene is long and the mRNA is susceptible to degradation or forms secondary structure during transcription, which leads to post-transcriptional instability in complete translation of the gene.
Mass Spectrometry Results
To verify whether the gene cluster was phenotypically successful in synthesizing dipeptide aldehydes, we used direct mass spectrometry to analyze the mass-to-charge ratio of dipeptide aldehydes (Figure 3-3-1).
We performed direct mass spectrometry on the wild-type strain and the +bgc33-sfp strain and analyzed the +bgc33-sfp strain according to a gradient of 100-fold dilution, 10-fold dilution of the solution and the stock solution. We did not find new peaks in the diluted liquid, but surprisingly, when we used the stock solution, we detected new peaks not found in the wild type. Although the amount was extremely low, we finally found the new product. Based on the results of real-time fluorescence quantitative PCR, we believed that the amount of products is greatly reduced under the influence of low transcription levels, and therefore we could only detect peaks with a very small area.
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