Team:NU Kazakhstan/Design

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Whole construct assembly design

Initially 4 transcriptional units were ordered from IDT company using iGEM promotion. These 4 units were designed (additional flanking 24 bp sequences) in a such way so that it could be amplified using PCR and appropriate primers (Figure 1).

Figure 1. Ordered sequences from IDT

List of designed primers

Fwd AphVIII (Primer 1) CTGGATTGGATGTGCTGTTGACCG

Rev AphVIII (Primer 2) GACAGTGGCTAATGGCTGAAACGC

Fwd ChrR (Primer 3) CTGGCAGGTACGATGTATCCTCGG

Rev ChrR (Primer 4) TGTAGGGAACTGGGTACGCAATCC

Fwd Chromodulin (Primer 5) CGTCAGGACACTTGTTGACTTGGC

Rev Chromodulin (Primer 6) TTGACCTGGTACGAAGTAGCCTGC

Fwd Supernova (Primer 7) AGATCCTAGTTTGACCTGCGTGCC

Rev Supernova (Primer 8) ATTGCTGGGAATCCTACGAGTCCG

As it can be seen, these 4 transcriptional units have overlapping regions of the length of 49 bp each for Gibson Assembly (Figure 2).

Figure 2. Gibson Assembly in silico (SnapGene)

Final construct looks like this (Figure 3):

Figure 3. Final construct

Design of primers for insertion into pChlamy_4 vector

pChlamy_4 vector ordered from company ThermoFisher was used to check presence of our proteins in C. reinhardtii by Western blotting. This vector is designed in a such way, so that after restriction-ligation cloning into multiple cloning sites, it is fused with resistance gene (Zeocin) and also has V5 epitope with 6xHis tag. During translation target protein is self-cleaved from resistance gene by FMDV 2A peptide. In order to clone our coding sequence into the pChlamy_4 vector, transcriptional units were PCR amplified using specially primers that added EcoRI from 5’ end and XbaI+PstI from 3’ end. In addition to this, start codon and stop codon were removed, because coding sequence was fused to the resistance gene and addition of additional protein tags require absence of stop codon. After successful PCR, obtained amplicon could be digested with EcoRI+XbaI for further protein analysis (Western Blot/protein purification). In case if protein is desired to be expressed without any additional tags, EcoRI+PstI cloning can be used (Figure 4).

Figure 4. Scheme for insertion into pChlamy_4 vector

Primers were carefully designed in such a way so that everything remained in frame (Figure 5):

Figure 5. Careful design of primers for insertion into multiple cloning sites of pChlamy_4 vector (example on SuperNova coding sequence).

Design of primers for CPEC (Circular Polymerase Extension Cloning) cloning of our parts into pSB1C3 plasmid

pSB1C3 linearized plasmid backbone was PCR amplified by using these two following primers:

Rwd pSB1C3, 39bp

tactagtagcggccgctgcagtccggcaaaaaagggcaa

Rev pSB1C3, 41bp

tctagaagcggccgcgaattccagaaatcatccttagcgaa

Each part was also PCR amplified in such a way so that it had flanking BioBrick Prefix and Suffix (Figure 6).
Following primers were used for each part (3 bp from 5’ end corresponding to pSB1C3 backbone, BioBrick prefix/suffix and 16-20 bp of overlapping nucleotides with target region at 3’ end of each primer):

Fwd ChrR, 43bp

ctggaattcgcggccgcttctagatgagcgagaagctgcaggt

Rev ChrR, 43bp

gactgcagcggccgctactagtattagatcttcacgcgctgaa

Fwd SuperNova, 43bp

ctggaattcgcggccgcttctagatgggtagcgaggtcggtcc

Rev SuperNova, 42bp

gactgcagcggccgctactagtattatcctcatcggacccga

Fwd promoter, 44bp

ctggaattcgcggccgcttctagagacggcggggagctcgctga

Rev promoter, 42bp

gactgcagcggccgctactagtctgcaaatggaaacggcgac

Fwd Chromodulin, 39bp

ctggaattcgcggccgcttctagatggaggaggaggagg

Rev Chromodulin, 39bp

gactgcagcggccgctactagtttaatcatcgccctcct

Fwd terminator, 44bp

ctggaattcgcggccgcttctagagctccgtgtaaatggaggcg

Rev terminator, 42bp

gactgcagcggccgctactagtgcttcaaatacgcccagccc

Figure 6. Circular Polymerase Extension Cloning scheme

After PCR purification of two amplicons (part and backbone), CPEC reaction was run. Then CPEC product was transformed into E. coli and plasmid extraction was performed.