Team:HKUST/Group1

Welcome

Chimeric Receptor Construction

The chimeric receptor expression cassette contains the N- and C- terminals of the rat OR RI7, flanking the TM2-TM7 ligand-binding domain of the c. elegans OR odr-10. The receptor sequence is first derived through fusion PCR, and then cloned into the yeast expression vector pESC-His for further localization and functional assay.

I. Primer DesignI.

We have designed several sets of primers for parts and BioBrick construction and DNA sequencing. The primer sequences are listed in Table 1. Primer statistics are calculated using NetPrimer.

For the chimeric receptor, primers are designed with 10 bp overlapping overhangs at the fusion junctions so that the fragments can anneal in fusion PCR. Two different reverse primers have been designed for the RI7 scaffold primers, one with stop codon incorporated into the sequence, and the other without. These two different alternatives can be chosen for construction of receptors with or without localization tags. In addition, nucleotide sequence in the primers has been modified in a few places to adjust for codon bias among c.elegans, rat and budding yeast.

The following diagram illustrates the schematic of the main primer design for the chimeric receptor.

Table 1 Primer sequences designed for the constructions

*Note:

1. Restriction sites are highlighted in blue.

2. Overhangs for fusion junctions are highlighted in red.

3. Stop codons

#Note:

For the fusion primers P3-P6, whole sequence Tm is indicated in black, while Tm for the main annealing part (without calculating overhang) is indicated in green.