Part:BBa_K3143006
pBAD-Cl2Y aaRS-pGlnS-Cl2Y aaRS-pProK-tRNA
Cl2Tyr ncAA system could incorporate 3,5-dichlorotyrosine non-canonical amino acid into protein of interst containing a TAG codon. It is an modified version originated from Peter Schultz's lab[1].
Figure 1: 3,5-dichlorotyrosine non-canonical amino acid chemical structure.
Usage and Biology
Cl2Tyr ncAA system could introduce 3,5-dichlorotyrosine specificity into the proteins.
In the 2019 BEAS_China design, we used this ncAA system to build a ncAA-dependent toxin-antitoxin system, see our Design.
Characterization
1. ncAA incorporation into GFP
In the non-canonical amino acid system, the aminoacyl tRNA synthetase is under the control of the arabinose promoter, while the tRNA recognizing the TAG codon is regulated by a constitutively expressed promoter.
Figure 2: Schematic illustration of genetic circuits for incorporation of 2 ncAAs into GFP(39TAG & 151TAG).
As can be seen from Fig 3, when arabinose or ncAA was not added to the system, the expression level of GFP was almost zero. When arabinose and ncAA was not added to the system, GFP was fully synthesized thus fluorescence signal can be detected.
Figure 3: Characterization of Cl2Y RS system.
2. ncAA-dependent Toxin-Antitoxin system
To better quantify the effect of insertion the TAG codon into Phd antitoxin, we first fused a GFP fluorescent protein using the GS Linker to the C-terminus of the PhD protein. By doing so, we can simply characterize the performance of the TAG codon insertion by measuring fluorescence.
Figure 4: Schematic illustration of genetic circuits for incorporation of ncAAs into Phd antitoxin.
First we tried to confirm that insertion of how many TAG codons could completely inhibit gene expression. We inserted 1-3 TAG codons just after the start codon, and then measured the fluorescence value of Phd-GS-sfGFP. The results showed the expression of the gene was well terminated in three designs, and the insertion of two TAG codons was the best.
Figure 5: Characterization of Phd-TAG-GS-sfGFP system
Next we attempted to introduce the ncAA system into the Phd-GS-sfGFP expression system with two TAG codons. When the ncAA system is expressed, we can see that the system that originally had no fluorescence produced a strong fluorescent signal. This shows that we have successfully established an Anticoxin (Phd) system that relies on ncAA. When ncAA is present in the system, Phd can be successfully expressed, and when ncAA is not present in the system, the expression of Phd is completely terminated.
Figure 6: Characterization of Phd(2TAG)-Doc system
Next, we inserted two TAG stop codons after the Phd gene start codon and transferred the corresponding Phd and Doc plasmid to C321 to construct the ncAA-dependent Phd-Doc Biocontainment System.
We then tested the system and the results are shown below: When the ncAA system is not OFF, there is no sign of any bacterial growth under the microscope. When the ncAA system is ON, bacterial’s growth is back to normal. This shows that the TA system we built that is dependent on ncAA system is successful. This system will ensure that NEZHA will get rid of security problems in practical applications.
Figure 7: A & B OD600 value and cell imaging for ncAA-dependent Phd-Doc Bioncontainment System after 24 hour's growth
Reference
1. Young T S, Ahmad I, Yin J A, et al.. An enhanced system for unnatural amino acid mutagenesis in E.coli[J]. Journal of molecular biology, 2010, 395(2): 361-374.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1221
Illegal BamHI site found at 1144 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 979
Illegal AgeI site found at 3701 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 2183
Illegal BsaI site found at 2361
Illegal SapI site found at 961
| None |