Team:HKUST/Group1

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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.

II. PCR

For optimized PCR efficiency and accuracy, Vent polymerase is chosen for amplification PCR reactions; KOD polymerase is used for fusion PCR. Gradient PCR is carried out for each step to optimize the reaction. The DNA fragments coding for the RI7 localization scaffold and the odr-10 ligand-binding pocket are first amplified separately from respective cDNA template (pHeI4 for RI7 and pPD9S.77 for odr-10) via PCR. These fragments are then fused together via fusion PCR.

In fusion PCR, reaction conditions and reactant concentrations must be carefully controlled to obtain satisfactory yields. DNA fragment fusion must be carried out in a stepwise process, by fusing RI7-N and odr-10 first (using P1 and P5), followed by annealing this fused sequence to the RI7-C fragment (using P1 and P2). PCR cleanup is essential in the procedures, preferably by gel purification, since residual primers may affect the reaction in the next step, leading to amplification of the original template instead of desired fusion reaction.

In the reaction, the two fragment templates are allowed to go through 1 to 2 PCR cycles without primers in order to create a fused template for amplification. The amplification primers are then added to the reaction mixture to allow for amplification of the whole fused sequence.

III. Cloning

After the chimeric receptor sequence is successfully derived through fusion PCR, standard cloning procedures are followed to construct a receptor expression cassette.

The pESC yeast epitope tagging vector pESC-HIS is used in the expression cassette construction. The chimeric receptor insert is cloned into MCS1 of the vector (under GAL10 promoter; with FLAG epitope tag) between the restriction sites EcoRI and NotI. The RI7 control construct can also be generated in the same way.

A GFP-tagged receptor can be generated by cloning the GFP tag into a constructed receptor expression vector, between the MCS1 sites of SpeI and SacI, replacing the FLAG epitope.