Team:BostonU/Parts
Part Submissions
Here is a link to our parts: Our parts page
We submitted the following three parts of our experiment to the iGEM registry:
As an iGEM team, we wanted to contribute novel and functional parts to the iGEM Registry.
We submitted 3 parts integral to our experiments as BioBricks. Here is a link to our parts page on the Registry. Below we describe these parts in more detail.
Recombination Directionality Factors:
orf7
This part (K1733000) contains the orf7 recombination directionality factor, corresponding 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 flanked by specific recombination sites.
Orf7 recognizes AttL and AttR recombination sites that flank sequences of interest, and, when both TP901-1 and orf7 are present, the sequence within the recombination sites can be manipulated. After performing the reaction, orf7 will no longer recognize the recombination sites, since they change to AttB and AttP sites, so the sequence cannot be reverted as such.
We characterized functionality of the intact orf7 against a fluorescent reporter plasmid. This plasmid encoded for an mRuby protein in the inverse orientation, between AttL and AttR sites. We transfected this reporter with an intact TP901-1 protein and the orf7 protein, and the proteins catalyzed the inversion reaction to yield expression of mRuby. Below is the characterization data of our orf7 part:
Dimerization Domains:
These parts (K1733001 and K1733002) contain the ABI (ABA insensititve 1) and PYL (pyrabactin resistance like) protein domains that dimerize in the presence of the small molecule abscisic acid (ABA). By fusing these dimerization domains to inert halves of a protein, and adding or removing ABA, we were able to control the function and activities of several split proteins. We believe that other iGEM teams can take advantage of this conditional dimerization system to regulate their own protein activities.
Both ABI and PYL are found in plants1. Thus, they can be implemented in mammalian cells, since the system is orthogonal. We tested our system using split integrase proteins in mammalian cells.
We characterized functionality of this domain in several split amino acid locations. One example shown below included splitting the TP901-1 protein and fusing halves to ABI and PYL respectively. We added ABA into our media to induce dimerization of the domains and protein halves, and measured the protein activity of TP901-1 afterwards. We were able to characterize some functional splits, since we regained TP901-1 activity after inducing dimerization. One functional split site (between amino acids 326-327) is shown below:
Recombination Directionality Factors:
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
- Liang, Fu-Sen, Ho, Wen Qi, Crabtree, Gerald R., “Engineering the ABA Stress Pathway for Regulation of Induced Proximity”, Sci Signal, 2011.