Team:Alma/Contribution
Our Contributions to the iGEM Registry
This year we sought to make contributions in two different ways. The first was to better characterize AHL synthase genes - these are genes for enzymes involved in quorum sensing and bacterial cell-to-cell communication. We initially thought these could be relevant to our project if we needed to control the population or activity of cells within the gut. This involved measuring both the activity (AHL production) and protein expression of several different cassettes. You can read more about this at the relevant registry pages; BBa_K204047, K574004, and K1692023 or see the description below.
The other major way we sought to make a contribution had to do with our assembly approach. While we relied on Gibson assembly for the initial steps, we wanted to use a modular Golden Gate assembly reaction for creating combination of the different genes involved in our pathway. Taking inspiration from the literature, we tested out an idea of adding Golden Gate sequences (BsaI sites and overhangs) to BioBricks in a universal way, so that modular assembly can be carried out using a small set of primers. This eliminates the need to create a separate primer set for every BioBrick that is used. This approach was tested out on several BioBricks, especially BBa_K515005, a GFP expression brick. A more completion description of this work is below, and a summary of the data is found on both the pSB1C3 part page and the BBa_K515005 part page.
Measuring Expression and Function of LuxI or LasI BioBricks
To test the expression and activity of BBa_K204047, K574004, and K1692023, we took the following steps. Equal volumes of mid-log cells were resuspended in fresh LB + Chloramphenicol. These cultures were incubated for 6 hours at 37C in a tube rotator. Cells were pelleted by centrifugation - the supernatant was saved for detection of AHL molecules (all those synthesized during the incubation).
From the remaining cell pellet, proteins were isolated using the B-PER reagent (Thermo-Scientific). Soluble and Insoluble proteins were then run on an SDS PAGE gel. The gel was stained with coomaise, and density analsyis was performed with ImageJ.
This is a representative image from several independent replicates. S = Soluble protein fraction, I = Insoluble protein fraction. The negative control used in this particular replicate was an uninduced culture of strain BBa_T9002.
This is a density analysis of the soluble lanes in the previous gel image; in particular, for the region between the 35 kDa marker and 15 kDa marker. (Blue line, left to right). The lines show a normalized density for each protein sample, and highlight that there are no significant peaks for the AHL synthase BioBrick cultures above background in the 20-25 kDa range, which is where the AHL synthases would be expected to be found.
From these results we were barely able to detect any expression of the AHL synthase genes - proteins such as LuxI and LasI should be present as bands slightly below the 25 kDa marker. However, the supernatants from these same cultures did display AHL when tested with the biosensor strain BBa_T9002 . In the graphs above, this shows various supernatants (from 40 minutes of AHL production to 2 hours) mixed with the biosensor strain - the fluorescence output and cell growth of the strain shows significant signal above the negative control. Thus, we conclude that the proteins are present but at very low quantities - likely a result of the degrons present, leading to a very short half-life.
At the same time as performing these experiments, we were able to detect protein expression for reporters such as RFP (BBa_J04450) and GFP (BBa_K515005), suggesting that our technique and experimental setup was not responsible for our inability to see expression of the AHL synthease genes.
Testing the performance of Universal Primers for Golden Gate cloning
Golden Gate assembly is a useful approach for modular combination of BioBricks. However, this type of assembly often requires separate primers be designed and ordered for each part to be assembled, even if the same overhang is used for several parts. This requirement is imposed in traditional PCR approaches because an extended region of the 3’ end of the primer is designed to match the unique sequence of a BioBrick.
We wanted to determine if Golden Gate overhangs could be added to BioBricks via PCR with universal primers – primers that will anneal to the Prefix and Suffix that is present on every BioBrick. This would enable a resource-limited lab, such as a High School iGEM team, to order a set of a few primers that can be applied to combine any BioBrick together. For high throughput assembly of different combinations of BioBricks, this would likewise greatly reduce the cost involved in primer design and synthesis.
Thankfully, there is precedence for the type of PCR result we wanted to achieve. In “One primer to rule them all: universal primer that adds BBa_B0034 ribosomal binding site to any coding standard 10 BioBrick.” by Bryksin et al, they designed primers in which a ribosome binding site was added using universal primers – they did this by including the new sequence in between two regions of primer annealing. Instead of a ribosome binding sequence, we created similar primers that include a BsaI site and a few different overhangs.
For example, the blue primer will amplify any protein coding BioBrick and add a BsaI site that will generate an AAGG overhang, while the suffix primer will add a BsaI site that adds a TGCA overhang (compatible with a PstI cut on pSB1C3). Such a fragment could be included in a Golden gate assembly with an appropriate Backbone and other fragments with overhangs. In our preliminary study, we choose enough overhangs to produce a 4 BioBrick + backbone assembly reaction. design and synthesis.
We wanted to not only use this approach to assemble parts in our project, but determine how robust this unusual PCR was, and how it can be used by other teams. Thus, we sought to characterize how well this worked with different primers and BioBricks. Our main set of experiments used BioBrick K515005, but this could be considered characterization of the backbone itself. This characterization was done by using different PCR cycling conditions, control primers, and ultimately sequencing some of the products.
To demonstrate this new approach, we desgined forward primers with overhangs sequences AATT, AAGG, ACTC, AGGT, and reverse primers with overhangs AAGG, ACTC, AGGT, and TGCA.
Using this (and other) BioBricks and the above primers, we carried out PCR reactions to determine if this approach was feasible. The reactions were generally setup as follows: 0.5uL of purified plasmid DNA for BioBrick BBa_K515005 was mixed with 9.5uL ddH20, 1.25uL of each primer (@ a 10uM concentration), and finally with 12.5uL of a 2X Mix for Q5 DNA Polymerase. Reaction conditions were setup as suggested by NEB for Q5 polymerase, typically with an annealing temperature between 51 and 55C. In some trials, 9uL of ddH20 and 0.5ul of DMSO were added. For a more complete description of these experiments and our attempts, visit the relevant section on the results page.
A representative gel is present on the left. PCR products from some reaction combinations were selected and sequenced from the middle towards the end - this way, we can confirm if the overhang and BsaI site were added correctly. VF2 and VR primers were used as controls in the event that some primer pairs gave no or incorrect products. The end of a sequencing run was determined by analyzing the chromatogram - an example of this analysis is below.
We had mixed results - some of these PCRs required optimization, and were sensitive to annealing temperature and the speed at which the temperature shifted (evident by comparing the reaction in an old thermocycler versus a new mini8 PCR machine). However, we were able to show that this approach can be viable and does indeed lead to products that should work in a Golden Gate assembly reaction. Our results for all pairs is represented below.
In the above table, green indicates a successfully confirmed reaction (with the bold cell representing the result corresponding to the representative chromatogram above), light green indicates a sequencing analysis with partial success/confirmation/matching, orange-pink (EtBr color) indicates a PCR reaction that gave the correct product, and purple indicates a reaction that did not work. Empty cells represent untested combinations. *These reactions did not work initially, but were successful at least once (required further optimization).