![]() ![]() Since reporter protein mRFP has color, we can easily intuitively find the optimal conditions through the change of color. BL21 strain containing toehold switch plasmid were cultured under different conditions. To increase the yielding of marker protein mRFP, some different culture conditions were optimized, including the pH value, temperature, fermentation time, and the concentration of transfected miRNA. However, after transfection with miRNA 221-3p into the BL21 strain transfected with pET-28a-toehold switch-mRFP, some transfected clones appeared red color, which were shown in Fig.4.įig.4 The effectiveness of toehold switch-mRFP.īacteria clones only transfected with toehold switch-mRFP appeared white color, while bacteria clones transfected with both toehold switch-mRFP and miRNA 221-3p appeared red color (miRNA 221-3p switched on the expression of mRFP). After it was transfected into BL21 strain, no mRFP protein (red color) could be observed with naked eyes, indicating that the toehold switch was effective. Toehold switch-mRFP was also cloned into pET-28a expression vector, constructing the recombined plasmid pET-28a-toehold switch-mRFP. The mechanism is shown as Fig.3.įig.3 The mechanism of toehold switch-mRFP. At the presence of miRNA 221-3p, it binds to its antisense sequence, opening the toehold switch to trigger the expression of mRFP, which is easily measured. It comprises the antisense sequence of miRNA 221-3p, RBS, Linker and part sequence of miRNA 221-3p, which form a toehold switch, as well as the gene of marker protein mRFP. Toehold switch-mRFP plasmid is designed to express the mRFP protein controlled by the toehold switch and miRNA 221-3p. M: Marker 1: PCR result Digestion result. Then it was identified as follows (Fig.2).įig.2 Identification of standard part pSB1C3-toehold switch-mRFP using PCR and digestion with EcoRI and PstI. To construct the standard part, toehold switch-mRFP was synthesized and checked the restriction enzyme information, which is shown as follows(Fig.1).įig.1 The map of toehold switch-mRFP described with SnapGene Viewer, showing the restriction enzyme information (no EcoRI and PstI sites).Īfter detecting the restriction enzyme information of toehold switch-mRFP using SnapGene software, it was inserted into the pSB1C3 plasmid to construct the standard part pSB1C3-toehold switch-mRFP with PCR method. It is used to detect the amount of miRNA 221-3p in samples. ![]() It was designed to express mRFP protein triggered by miRNA 221-3p. Both of them worked as expected:īBa_K4167001 part is a standard part, in which toehold switch-mRFP sequence was inserted into pSB1C3 plasmid. *.zip, *.7z, *.gzip, *.gz, *.bz2, *.tar, *.In this part, we want to show engineering success by demonstration the design and construction of 2 new parts. Table 1: File Types Imported by SeqMan NGen for De Novo Assemblies and Templated Alignments (Special Workflows) Note: SeqMan NGen supports the export of data in. Below are the file types supported for import for both normal workflows and special workflows. SeqMan NGen is included within Lasergene Genomics. Image Files (*.png, *.jpg, *.gif, *.bmp 8)ġ File-Save will save back as original file type. These files are not supported through the menu system.ĩ Projects or files must contain protein sequence data.ġ0 Only the sequence, features and comments of the file will be read in.ġ3 Lasergene does not support ABI files without base calls.ġ4 Protean 3D supports version 3.2 of PDB files.ġ5 Available to support gap closure in SeqMan Ultra and for assembly in SeqMan NGen.ġ6 Supports *.txt and *.gp UniProt files only.įASTA Format (*.fas, *.fap, *.fna, *.fasta) All other files will be ignored.Ģ Multiple Lasergene DNA sequence files can be created by EditSeq, SeqMan Pro, and MegAlign.ģ FOF can not contain reference to another multiple sequence file format such as FastA, DNA* mseq, or Zip.ĥ Referred to collectively as “Lasergene Documents” in “Files of Type” or “Show pull-down menus”.ħ GenBank formatted features will be parsed.Ĩ Through “drag and drop” only. SeqMan NGen Transcriptome Files (*.transcriptome)ġ Only zip files containing ABI, SCF3, Lasergene protein or Lasergene DNA files. SeqMan NGen Assembly Projects (*.assembly) Protein Data Bank files (*.pdb, *.ent, *.pdb.gz, *.ent.gz, *.zip, *.txt) Imported File Types File FormatĤ54 Life Sciences output files (*.sff *.fas and *.qual)įASTA Format (*.fasta, *.fas, *.fap, *.nt, *.aa)įASTA Alignment with Gaps (*.fasta, *.fa, *.fas, *.fna, *.fap, *.nt, *.aa, *.frn, *.faa, *.ffn, *.mfa) Lasergene Molecular Biology and Lasergene Proteinīelow are the file formats supported for import and export by the applications within the Lasergene Molecular Biology and Lasergene Protein packages. ![]()
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