Team:GA State SW Jiaotong/Basic Parts
Overview
This year, we designed five new parts and used twenty-four basic parts in our project. In order to construct the new parts, different approaches including direct order from IDT and Genscript, enzyme digest, ligation, and Gibson assembled were applied. From IDT and Genscript, we obtained Gas6 sequences codon-optimized for Pichia pastoris, EGF, sok, and hok coding sequences codon-optimized for Bacillus subtilis. During our collaboration with our partnership team, ShanghaiTech_China, we obtained the dual regulation system in pET28a-TRP plasmid. Other parts were obtained by enzyme digest and ligation, PCR amplification, as well as Gibson Assembly from Escherichia coli BL21 (DE3) and Bacillus subtilis WB600.
New Parts
| Name | Type | Description | Lector | length |
|---|---|---|---|---|
| BBa_K3842000 | Coding | Coding sequence for growth aresst-specific protein | Xiaoyi Feng | 2069 |
| BBa_K3842007 | Coding | Epidermal growth factor | Xiaoyi Feng | 3498 |
| BBa_K3842016 | Coding | Coding sequence for suppression of killing RNA antitoxin | Xiaoyi Feng | 66 |
| BBa_K3842017 | Coding | Coding sequence for host killing toxin | Xiaoyi Feng | 247 |
| BBa_K3842021 | Coding | TRP coding sequence | Xiaoyi Feng | 327 |
Gas6 Coding Sequence
Gas6 refers to the growth arrest-specific protein 6. The amino acid sequence of Gas6 we used for our project came from Homo sapiens. However, due to the codon bias, DNA sequence of Gas6 protein needed to be optimized for efficient expression in Pichia pastoris. As shown in Fig. 1 below, codon quality was improved significantly after codon optimization. Also, signal peptide for Pichia pastoris was added to the beginning of the coding sequence for direct secretion of gas6 from cytoplasm. 6xHis tag was added to the end of the coding sequence for easier purification of Gas6 protein from the yeast solution.
Fig. 1. Codon quality plot. This plot shows the codon quality before and after codon optimization of the Gas6 coding sequence.
Figure 2. DNA sequence map of pPICZαA-Gas6 plasmid.
EGF Coding Sequence
EGF refers to the human epidermal growth factor. Amino acid sequence of EGF came from Homo sapiens, which can be applied in treating human diseases. Considering the codon bias, DNA sequence of EGF protein needed to be optimized for expression in Bacillus subtilis. Also, one trail of EGF coding sequence were ligated with 6xHis tag for facilitate the purification of EGF.
Figure 3. DNA sequence map of pNW33N-EGF plasmid.
Sok/Hok Suicide System
Our suicide system part is based on the Sok/Hok toxin-antitoxin system. This part contains coding sequence of toxin protein Hok and an antisense RNA of hok transcript, Sok. In our design, the transcription of Sok is controlled by a lactose promotor while that of hok is inhibited by a tryptophan promotor. In the suicide system, DNA sequences of trp repressor and Lac I repressor will be introduced. With the existence of lactose and tryptophan in the solution, Lac I repressor can’t bind with lac operator. Thus, sok coding sequence regulated by Plac can be transcribed normally, while the transcription of hok will be blocked. On the other hand, without lactose and tryptophan in the system, bacteria die due to the cell membrane depolarization caused by Hok toxin.
Figure 4. Map of Sok coding sequence with homologous arm.
Figure 5. Map of Sok coding sequence with homologous arm.
Figure 6. DNA sequence map of suicide switch combining Sok/Hok with dual regulation system.
