Introduction
On this page, we present our contributions to the iGEM community. In our project, we have developed several skills, and we hope to pass them on to the future generations of the iGEM teams. If their designs also contain reactions of nucleic acids, what we have done may bring them choices or inspirations. We hope that our project can help the teams that choose the diagnostic track in the future. If we can also help and influence the teams on other tracks, that would be great.
Parts
Our team contributed to the iGEM Registry through adding new components. We hope to use Klenow-SH3 [BBa_K3894022] 5'-3' polymerization and strand displacement activities, but wipe off its 3'-5' exonuclease activity. Therefore, through the mutation of D424A, we generated a polymerase that was deficient of the exonuclease activity: Klenow.mut-SH3 [BBa_K3894012]. We constructed an engineering plasmid and successfully expressed the Klenow.mut-SH3. In our project, it is used for single-stranded DNA (ssDNA) replacement, and these point mutations protect our ssDNA from being degraded. Therefore, if future teams have similar application scenarios like ours, they can consider using this new part provided by us.
We also added other new components. BbvCI-R1[BBa_K3894017] and Nb.BbvCI-R2 [BBa_K3894018] are the two heterologous subunits of the restriction enzyme R.BbvCI. The recognition sequence of the restriction endonuclease BbvCI from Bacillus brevis is CC^TCAGC/GC^TGAGG. The R1 subunit will cleave the DNA to generate a nick. The R2 subunit cleaves the top strand CC^TCAGC. We mutated the E177G site of BbvCI-R2 to generate a mutant that only recognizes and binds the target sequence, but does not cleave it when forming a dimeric enzyme. We hope to provide reference work and experience to the subsequent iGEM teams for their selection of nickases and enzymes with strand displacement activity and combined in vitro through our experiments.
Figure 1. The positive transformation result of Klenow.mut-SH3.
CRISPR Guideline
Our team used the CRISPR/nCas9 this year for the design of CRISPR. Regarding the problems encountered in the design and experimental process, we attended an online CRISPR conference, and communicated with other iGEM teams that used the CRISPR technology. The CRISPR Guideline may help the future iGEM teams when using the CRISPR technology. Click [Collaboration] page to learn more about the CRISPR conference.
Hardware
Our hardware part is mainly composed of a heat preservation device that can be used for the extraction of nucleic acid and storage of reaction solutions. Our whole device is easy to be used in detecting color changes. It is light, portable, and easy to be transported, which greatly reduces the test cost. For the popularization of our method, the device can be used at home and different outdoor scenes. Our heat preservation device can provide a suitable temperature for the reaction solution, and can also implement a high-temperature inactivation state after a nucleic test is completed, which prevents potential contamination to the environment. We hope our design can provide inspiration to the subsequent iGEM teams that need to use different reaction conditions in their projects.
Figure 2. The hardware designed by us.
