Team:BostonU/Parts
Part Submissions
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:
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:
Importantly, our part was characterized in a mammalian expression system. We have not characterized this part in other chassis.
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 plants. 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:
In the above experiment, we tested both domain fusion orientations. Proper TP901-1 activity led to expression of our mRuby fluorescent protein, which can be seen in both cases above. Read more about our fluorescent reporter experiment here.
Considerations with other parts: We intended to submit more parts to the iGEM registry, including other dimerization domains that we used (FKBP and FRB, CRY2 and CIBN), other integrases and RDFs (TP901-1, PhiC31, and gp3). Some of these were already in the Registry. However several of these protein domains were extremely large and did not conform with the BioBrick standard since they contained several internal restriction sites.
Considerations with other parts
We intended to submit more parts to the iGEM registry, including other dimerization domains that we used (FKBP and FRB, CRY2 and CIBN), other integrases and RDFs (TP901-1, PhiC31, and gp3). Some of these were already in the Registry. However several of these protein domains were extremely large and did not conform with the BioBrick standard since they contained several internal restriction sites.
We were able to introduce a silent mutation into ABI such that it was BioBrick compatible (thanks to IDT for the synthesis offer). We were also easily able to submit orf7 and PYL because these were BioBrick compatible. We recognize that working with large parts pose such BioBrick issues, and we discuss possible ways to mitigate this on our mammalian synthetic biology challenges page.
We also intended to characterize the spCas9 part from the 2013 Freiburg team using flow cytometry, in part to also have an intact spCas9 control for our own experiments. This part was a bacterial-optimized part; we aimed to add a Nuclear Localization Sequence (NLS) to the spCas9 protein such that it would function better in our mammalian system, as the protein needs to be in the nucleus to have proper functionality. However, we had difficulties cloning this part, and furthermore, found it difficult to re-BioBrick.
Citations
- Liang, Fu-Sen, Ho, Wen Qi, Crabtree, Gerald R., “Engineering the ABA Stress Pathway for Regulation of Induced Proximity”, Sci Signal, 2011.