Team:CCU Taiwan/Safety

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Positive & negative
controls E. coli
(DH5α and BL21-DE3)

It is important for everyone to protect the ecosystem, specifically when people try to improve life through scientific experiments. Safety procedures should be set up for each experiment, and team members should perform experiments following the safety guidelines. Therefore, all team members had to pass the laboratory safety education training held by CCU, and we always comply with our lab safety rules.

Positive and negative controls

To examine the activity and specificity of our CRISPR-Cas12a system in detecting the ASFV genome sequence, we designed six positive and six negative control vectors. The positive control vectors harbor a partial DNA sequence of the ASFV p72 gene, while the negative control vectors harbor the corresponding sequence with a mutation. The partial sequence of p72 gene in the positive control vectors is about 398 bp in length, and shows no obvious evidence for coding an open reading frame. The difference between positive and negative control vectors is that, the first 5 base pairs of the CRISPR-Cas12a targeted sequence are mutated in the corresponding negative control vector. We received approval for this by submitting the iGEM check-in form, and we ensured that all of the target DNA sequences are from part of a non-infectious protein gene (p72 gene) and cannot express any full-length peptides.

E. coli (DH5α and BL21-DE3)

E. coli strain DH5α and ampicillin were used to amplify LbCas12a expression vector (modified from pMBP-LbCas12a H759A vector; addgene #113518), TA vector encoded DNA template for crRNAs, and positive/negative control vectors for LbCas12a activity assay. E. coli strain BL21-DE3 was used to produce LbCas12a protein.

The discarded culture medium (Terrific Broth) was processed with sodium hypochlorite to avoid the spread of ampicillin-resistant E. coli . All the equipment and disposables used in the experiments were sterilized by autoclave or 75% EtOH. Then, a professional from the CCU environmental safety center managed the final processing of the experimental waste.