Team:BostonU/Parts

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Parts Submissions

Here is a link to our parts: Our parts page

We submitted the following three parts of our experiment to the iGEM registry:

Dimerization Domains:

ABI and PYL

These parts contain the ABI (ABA insensitive 1) and PYL (pyrabactin resistance like) protein domains that will dimerize with each other in the presence of abscisic acid. By fusing these dimerization domains to inert halves of a protein, we were able to control protein function and induce dimerization, and protein activity, with the addition of ABA (abscisic acid). We were therefore able to regulate protein activity by taking advantage of these dimerization domains.

Both ABI and PYL are found in plants¹. Thus, it can be tested in mammalian cells, as the ABI-PYL-ABA system is completely orthogonal. We tested this system with our split integrases in mammalian cells.

We used this domain in our experiment to construct split protein complexes. We split the TP901-1 protein and fused the halves to ABI and PYL. We then added abscisic acid into the system to induce dimerization of the domains and the protein halves, and measured the protein activity TP901-1 afterwards. We were able to characterize some split sites as functional, as we regained the TP901-1 activity after inducing dimerization. One functional split site is shown below:

Here, we split the TP901-1 protein between amino acids 326 and 327. We fused the halves to ABI and PYL, and also tested the fusion in reverse orientation. Proper TP901-1 activity would lead to expression of a fluorescent protein. This graph shows that we were able to regain some TP901-1 activity, so we were able to validate this split site as functional.

Recombination Directionality Factors:

orf7

This part contains orf7, which is the corresponding recombination directionality factor to the TP901-1 integrase. It catalyzes the unidirectional inverse reaction of the TP901-1 integrase, allowing for the inversion, deletion, and cassette exchange of sequences of DNA.

Orf7 recognizes AttL and AttR recombination sites that flank some sequence of interest, and, when both TP901-1 and orf7 are present, the sequence within the recombination sites is can be inverted, deleted, or undergo cassette exchange with another sequence of DNA. After performing the reaction, orf7 will no longer recognize the recombination sites (AttP and AttB), so the sequence will not continually inverting.

To test this part within our project, we used a reporter that contained a gene that codes for a fluorescent protein flanked by the AttL and AttR recombination sites. Originally, this fluorescent protein is unexpressed, but when we add TP901-1 and orf7 to the system, the proteins catalyze the inversion reaction, the sequence in between the recombination sites is flipped, and the fluorescent protein is expressed. Below is the fluorescence expressed for the full orf7 RDF.

Orf7 and TP901-1 can be used in further applications to create a switch to flip and knock out a gene of interest.

We had intended to submit more parts, including the other dimerization domains that we'd used (FKBP/FRB and CRY2/CIBN) and the other integrases and RDFs that we had split (TP901-1, Phic31, and gp3). However, these protein domains were large and were not up to the Biobrick standard, containing at least one of the restriction sites within the protein sequence. Often these proteins in mammalian cells do not conform to the Biobrick restrictions. Luckily, orf7 and PYL were up to Biobrick standards. ABI had one restriction site, so we had to introduce a silent mutation and had IDT synthesize a new part for us to Biobrick. We recognize that working with mammalian parts often leads to problems with Biobricking, and so we have proposed some solutions to this issue on our Mammalian Syn Bio Research solutions page here.

Additionally, we had intended to characterize an SpCas9 part from the 2013 Freiburg team using flow cytometry. We had wanted to add an NLS (nuclear localization sequence) to the part in order to improve its function. The NLS tags the SpCas9 for transport into the nucleus, and since SpCas9 performs its function into the nucleus, this NLS would allow for optimal SpCas9 activity. However, this part was difficult to clone. We wanted to characterize and submit our human codon optimized SpCas9 with an NLS; however, this was not up to Biobrick standards.

Citations

1. Liang, Fu-Sen, Ho, Wen Qi, Crabtree, Gerald R., “Engineering the ABA Stress Pathway for Regulation of Induced Proximity”, Sci Signal, 2011.