Team:UCSC/Contribution
Contributions
Navigating Plasmid Incompatibility
During the course of our project, we encountered several difficulties that taught us important lessons not only about working on our specific project, but also about conducting synthetic biology research. We want to share the lessons we learned from our mistakes so that future iGEM teams and/or other synthetic biologists do not make the same mistakes!
Our most significant difficulty stemmed from plasmid incompatibility. The final step in Clonetegration (the method we used to engineer our mcherry-integrated E. coli model as described in our
We were able to integrate the pOSIP-KL-mcherry plasmid into the chromosome of C41 pLysS, but all attempted pE-FLP transformations failed. We explored the inability of our C41 pLysS-pOSIP integrants to take up pE-FLP by transforming native, known-competent C41 pLysS cells with pE-FLP. We observed no colonies for both pOSIP integrants and native, competent C41 pLysS cells, indicating that the C41 pLysS strain is unable to take up the pE-FLP plasmid. We determined that incompatibility between the p15A ori on the pLysS plasmid and the pSC101 ori on the pE-FLP plasmid contributed to the C41 cells’ inability to take up the plasmid [1]. To avoid this issue, we switched strains to DH5α E. coli (NEB) which do not contain any native plasmids. After performing the initial integration of pOSIP-KL-mcherry into NEB DH5α, we were able to transform pE-FLP and verify the excision of the integration module from the chromosome(as seen in
We learned the importance of checking individual origins of replication for incompatibility when working with multiple plasmids in a single chassis. To help other teams avoid this problem in the future, we made a visual representation of plasmid incompatibility groups for reference when using multiple plasmids at once.
A visualized table for determining incompatibility between different plasmid incompatibility groups. The pMB1 replicon contains pUC vectors, and pBR322 and its derivatives. The p15A replicon contains pACYC and its derivatives. This table was adapted from OpenWetWare [2 ].
Parts
Progenie utilizes the INTEGRATE system, a CRISPR RNA-guided DNA integration system presented by Vo et al. in 2021 [1]. We reached out to Leo Vo at Columbia University [1] and received the pSPAIN plasmid as a gift to work with during our project. Here, we propose to add the pSL1142 (pSPIN, pBBR1 backbone) [3] plasmid to the iGEM parts registry. Along with its backbone (pBBR1 ori and kananycin resistance), this pSPIN plasmid contains the INTEGRATE cassette: J23119 constitutive promoter (Part:BBa_J23119), the V. cholerae CRISPR array, TniQ, Cas8, Cas7, and Cas6 (TniQ-Cascade complex), TnsA, TnsB, and TnsC (transposase elements), and finally, the donor DNA region flanked with transposon L and R ends. The guide-RNA spacer in this construct is a placeholder that contains BsaI sites for easy Type IIS restriction enzyme cloning. This part has not yet been added to the iGEM registry, and will help future teams working with bacterial genome engineering.
During the course of our project there were a number of genetic elements utilized/developed for our gene elimination mechanism, detection system, and partnership. To support the simplicity of using Golden Gate assembly we worked to develop a number of parts that contain type IIS sites. Down below is a table containing parts and general information.
| Part Name | Part Number | Part Type | Description | Designer | Length |
|---|---|---|---|---|---|
| pET-52b(+) backbone | BBa_K3808001 | Coding region | Protein expression | Rose Delvillar | 5.2 kb |
| pET-52(+)backbone + ccdB insert | BBa_K3808010 | Coding region | Protein expression | Rose Devillar, Franklin Zheng, Tanya Ivanov, and David Kelaita | 5.6 kb |
| INTEGRATE (pSPIN from addgene) | BBa_K3808004 | Coding region | RNA-guided transposase system | Leo Vo et al. | 14 kb |
| pSPIN F primer | BBa_K3808008 | Coding region | Amplifies the CRISPR array of interest | David Kelaita | 18 bp |
| pSPIN R primer | BBa_K3808009 | Coding region | Amplifies the CRISPR array | David Kelaita | 23 bp |
