Extending the knowledge of existing parts and adding new parts gives iGEM teams opportunities to step back from their project and expand their views on topics from other areas of knowledge. This year we were very excited to try doing both!
http://parts.igem.org/Part:BBa_I712074
In 2007, iGEM07 Ljubljana contributed to a part of specific and strong T7 promoter from T7 bacteriophages. We were interested to investigate how the research has progressed on this topic.
Numerous studies have tried to stabilize the expression of the T7 promoter by T7 phage polymerase but there was no success in improving the gradual decline of target protein expression [1]. Moreover, despite the highly specific mechanism of how T7 polymerase binds the promoter during the transcription initiation, it is unknown to what extent sequences in the extended initiation bubble impact on transcription [2].
Research published by Microbial Cell Factories highlights that the major contributing factor to decrease of gene expression by the described T7 system is a random chromosomal mutation [1]. Moreover, this mutation occurs in the sequence encoding the T7 phage RNA polymerase, which leads to non-overproducing cells to dominate the culture. Therefore, researchers tackled this issue by developing a method to eliminate non-overproducing cells from the culture [1]. In order to achieve low gene expression, a stable T7 system was created with dysfunctional T7 RNA polymerase. This goal was achieved by maintaining only target protein-expressing cells in the culture leading to a stable high level of recombinant protein expression [1].
Another research tackled the same issue from yet another perspective. It has been shown that bacteriophage T7 lysozyme is a inhibitor of T7 RNA polymerase which naturally lowers gene activity [3]. In a paper published by Journal of Molecular Biology, it has been shown that if low levels of T7 lysozyme are supplied by plasmid they are sufficient to increase the target protein production upon the induction of T7 RNA polymerase [4]. This induces the activity of T7 RNA polymerase allowing further production of protein [4].
This was our attempt to recreate the EPFL 2019 igem team’s PVA microneedle patch for DNA extraction. They ordered a PDMS mold inside which they put a 10% wt PVA solution to create the MN patch. We attempted to complete this entire process in-house in the following way. We believe that with this contribution a lot more teams can attempt to recreate and optimize a novel DNA extraction method which was first presented by the EPFL 2019 igem team.
We first designed a the mold and found the negative of it such that the printed file will look like the microneedle patch
We then made the PDMS solution for the mold in a 1:10 ratio (Curing agent:PDMS)and inverted the printed product into it
We left it in an oven at 65 degrees celsius to cure
Once it was ready we removed the printed product carefully - ensuring the mold is not damaged.
http://parts.igem.org/Part:BBa_J61004
pAC-TetInv is a medium copy plasmid under the control of a tet promoter.
The first step in the construction of the plasmid pAC-TetInv consists in PCR amplifying the inv gene (GenBank accession no. M17448) from Y. pseudotuberculosisgenomic DNA with oligonucleotides ca783F (5'-CTGAAGGATCCGTTTGACGTATGACAGGTATGC-3') and Rinv (5'-AGGGTCGAATTCTTATATTGACAGCGCACAGA-3'). In order for these two enzymes to be inserted into similar sites of Pac581, they need to be digest with BamHI and EcoRI, The components of the plasmid are mainly a TmBterimnato derived from pBAD/Myc-HisA, atetpromoter, a chloramphenicol resistance gene, and most importantly a p15A origin of replication.
Regarding the lux operon, it was assembled from plasmids pKE705 and pKE555 by first using Xhol and Xbal for digestion then by ligating the e580 bp fragment to a 2980 bp XbaI/BglII fragment of pKE555. PCR with oligonucleotides ca742F (5'-CAGTCGGATCCTTAATTTTTAAAGTATGGGCAATC-3') and ca721R (5'-CACTGGAATTCGTAATGACAGATAATTTTACTC-3') was used to amplify the ligation product. Moreover, BamHI and Ecori enabled the digestion of the full-length of lux operon gene enabling it to be inserted into similar sites of pPROBE-gfp[LAA]53. In consequence, this Probe-lux results in plasmid pPROBE-luxIR. PCR amplification of the lux operon in pPROBE-luxIR with oligonucleotides ca837F(5'-GGTATGCGGCCGCTTAATTTTTAAAGTATGGGCAATC-3') and ca837R (5'-CACTTGGATCCGTAATGACAGATAATTTTACTC-3') and then the insertion of this operon into the NotI and BamHI sites of pAC-TetInv results in the construction of the plasmid pAC-Lux Inv. This method enables a replacement of the tet promoter with the lux operon. Finally, the last plasmid that was constructed was Plasmid pAC-LuxGfp. PCR amplification was carried on the luxand gfp genes of plasmid pPROBE-luxIR through the oligonucleotides ca837F and ca803R (5'-GCAACGGTCTCGAATTCCCTTAGCTCCTGAAAATCTCGC-3'). NotI and BsaI, served as the digestion enzymes to enable its insertion into the NotIand EcoRI sites of plasmid pAC-TetInv.
The function of this plasmid was to constitutively express the invasion protein. Anchored in the outer membrane, invasin is a long rigid protein that extends 18 nm from the bacterial cell surface. This protein enables the internalization of bacteria into mammalian cells namely tumour cells including gepithelial, hepatocarcinoma, and osteosarcomaline, without any additional known adhesion molecules. However, bacterial internalization is only possible in particular cell density, hypoxia, and inducible inputs. In fact, engineered bacteri are non-invasive under conditions of cell-density and normal aerobic growth,. Sensors are only activated above a critical cell density or in a hypoxic environment resulting in the synthesis of invasin fromY. Pseudotuberculosis followed by the invasion of HeLa cells.
Reference for BBa_J61004
[1] Kesik-Brodacka, M., Romanik, A., Mikiewicz-Sygula, D., Plucienniczak, G., & Plucienniczak, A. (2012). A novel system for stable, high-level expression from the T7 promoter. Microbial cell factories, 11(1), 1-7. [2]Conrad, T., Plumbom, I., Alcobendas, M., Vidal, R., & Sauer, S. (2020). Maximizing transcription of nucleic acids with efficient T7 promoters. Communications biology, 3(1), 1-8. [3]Zhang, X., & Studier, F. W. (1997). Mechanism of inhibition of bacteriophage T7 RNA polymerase by T7 lysozyme. Journal of molecular biology, 269(1), 10-27. [4] Studier, F. W. (1991). Use of bacteriophage T7 lysozyme to improve an inducible T7 expression system. Journal of molecular biology, 219(1), 37-44.
Anderson, J. C., Clarke, E. J., Arkin, A. P., & Voigt, C. A. (2006). Environmentally controlled invasion of cancer cells by engineered bacteria. Journal of molecular biology, 355(4), 619-627.
