Contribution | Stony-Brook

Parts

New Basic Parts

Part Name	Type	Description
BBa_K5090000	CDS	Argonaute2, which is codon optimized and has a GST tag to express in E. coli or E. coli-based cell-free systems.
BBa_K5090004	Regulatory	P2A, a "Self cleaving peptide" that allows for the simultaneous expression of multiple proteins from a single RBS and start codon.
BBa_K5090001	CDS	LacI, a transcriptional repressor that binds DNA at the site of lac operators. This specific version was enhanced by Laird et al. to be more effective than wild-type LacI.
BBa_K5090005	Regulatory	P2A, "Self cleaving peptide" that allows for the simultaneous expression of multiple proteins from a single RBS and start codon.
BBa_K5090003	CDS	GFP, a fluorescent protein used as a reporter to indicate gene expression.
BBa_K5090002	CDS	L7Ae, a translational repressor that binds DNA at the site of kink turns.
BBa_K5090006	Regulatory	The reverse-complement of human miRNA-326 (hsa-miR-326), serving as a target site for Argonaute2 when miRNA-326 and the gene to which the target site is attached has been transcribed.
BBa_K5090010	RBS	Combination of BBa_J61100 (an Anderson RBS) and the complementary target site for miRNA-326, meant to prevent mRNA translation if microRNA-326 and Argonaute2 are present.

New Composite Parts

Part Name	Type	Description
BBa_K5090007	Device	Implementation of the "dual regulation system" created by Wang et al., with transcriptional repressor LacI and translational repressor L7Ae for binding with lac operators on DNA and kink turns on mRNA respectively, with the complementary target site for microRNA-326 present within the open reading frame of LacI, and with P2A allowing for LacI and L7Ae to be expressed under the same promoter and RBS while nonetheless being created as separate proteins and for the amino acids coded for by the target site for miRNA-326 to be separated from LacI so as to avoid interference with its function.
BBa_K5090008	Device	Implementation of Codon Optimized GST-Ago2 such that it binds to and cleaves target sites on mRNA strands complementary to the microRNA strands Ago2 loads.
BBa_K5090009	Device	Implementation of GFP under regulation of two lac operators (lacO2) and a kink turn such that its fluorescence is inhibited by LacI and/or L7Ae when present.
BBa_K5090011	Device	Implementation of the "dual regulation system" created by Wang et al., with transcriptional repressor LacI and translational repressor L7Ae for binding with lac operators on DNA and kink turns on mRNA respectively, with the complementary target site for microRNA-326 present within the open reading frame of LacI, and with P2A allowing for LacI and L7Ae to be expressed under the same promoter and RBS while nonetheless being created as separate proteins.
BBa_K5090012	Device	Implementation of the "dual regulation system" created by Wang et al., with transcriptional repressor LacI and translational repressor L7Ae for binding with lac operators on DNA and kink turns on mRNA respectively, with the complementary target site for microRNA-326 present after the RBS but before the open reading frame for LacI and L7Ae, and with P2A allowing for LacI and L7Ae to be expressed under the same promoter and RBS while nonetheless being created as separate proteins.

As our results demonstrate and our implementation page discusses, an implementation of these parts could be used as part of an accessible and affordable assay that would detect significant markers of B-cell lymphoma sooner than traditional diagnostic methods. This screening would get patients on the path to being tested by more traditional sooner than currently occurs, which is typically not until patients have developed symptoms and lymphoma has become harder to treat.

Design Considerations

Our team took several measures to ensure the safety of our team members and other colleagues at Stony Brook University while conducting our experiments.

In-Vivo

In an in-vivo system using bacteria, expressing eukaryotic proteins like Ago-2 could be quite challenging since it could often require codons that are not typically used in bacteria. To address this problem, Rosetta E. coli, a derivative strain of BL21, could be used as the experimental organism. This strain contains several genes coding for tRNAs that carry codons rarely found in bacteria like AGG, AGA, AUA, CUA, CCC, and GGA via the pRARE plasmid with a chloramphenicol resistance selection marker (“RosettaTM(DE3) Competent Cells”; Waegeman and Soetaert, 2011).

While seemingly promising, for our project we had to consider another factor: hydrolytic enzymes, specifically, ribonucleases (RNases). While the function of RNases was not limited to the degradation of RNAs, this was a point of concern because we needed to address the problem of possible miRNA-326 degradation via RNases upon entry inside the bacterial cell (Bechhofer and Deutscher, 2019). As a result, using RNase-deficient bacterial strains was brought forth as a possible solution. In particular, the MRE600 strain, lacking in RNase I activity, was a commonly used bacterial strain when conducting experiments involving various RNAs (Kurylo et al. 2016). Since MRE600 had minimal RNase I activity, we would be able to resolve the concern of miRNA-326 being degraded when entering the cells. It is also interesting to note that the lack of RNase I activity does not seem to pose additional harm to the bacteria, but still allows normal metabolic processes to occur (Bechhofer and Deutscher, 2019).

With this in mind, if we would like to proceed with using the MRE600 strain as our experimental organism, we would also then circle back to the very first issue of answering the question of how to actually express the eukaryotic protein, Ago-2, in bacteria. The next option, besides using the Rosetta strain, was codon optimization. Each species has a preference for what codons they choose to use, even if the codons are all encoding the same amino acids. This is known as the codon usage bias, which is important because the codon appearance frequency positively correlates with the concentration of tRNAs that carry the amino acids (Fu et al., 2020). So if a bacteria is transformed using a non-codon optimized Ago-2 gene sequence, very likely, there will be eukaryotic codons in that sequence that are rarely used by bacteria, and when this happens, the protein synthesis efficiency decreases since there will be very low concentrations of the rare-codon carrying-tRNAs within the bacterial genome (Fu et al., 2020). To maximize translation efficiency and expression, the Ago-2 gene sequence was optimized by replacing the rare eukaryotic codons with more commonly used bacterial codons via the IDT codon optimization tool (“Codon Optimization Tool”).

miRNA Heat Shock Assay

For future iGEM teams that wish to design and engineer bacterial miRNA detectors, we have collected preliminary evidence that strongly suggests bacteria can be successfully transfected with miRNA. Additionally, we had provided access to our miRNA heat shock assay protocol as a resource. Briefly, in our experiment, we tagged miRNA-326 molecules with fluorophore (i.e., FAM), so upon successful miRNA transfection into bacteria, confocal microscopy could be performed to compare the fluorescence levels for bacterial samples with or without miRNA transfection, in which bacterial samples with miRNA transfection would have a higher level of fluorescence.

We would like to add that other teams in the past have worked with bacterial miRNA detectors like the 2022 ICJFLS iGEM team who designed miRNA toehold switches that were then transfected into bacteria (“Results”, 2022). However, in contrast to our project, based on the information provided on their Wiki, it seems that no formal assays have been performed to confirm the efficiency at which bacteria had successfully uptaken the miRNA.

Growth Condition Optimizations for Endogenous Bacterial Promoter Expression

For future iGEM teams looking to express recombinant proteins (e.g., Ago-2) in bacteria under endogenous e.coli promoters, it is important to take into account the possibility of inclusion of body formations. Inclusion bodies are soluble or insoluble macromolecule aggregates, typically misfolded non-functional proteins, found within the cell that is formed when the protein homeostasis is disrupted (de Marco et. al 2005, Bhatwa et al. 2021). Oftentimes, the appearance of inclusion bodies is associated with stress or disease both for a cell or for an individual, in which on a macro-scale, for example, high concentrations of misfolded protein aggregates can cause Alzheimer's disease (Bhatwa et al. 2021). For our project, we had to opt for endogenous E. coli promoters because promoters such as T7 require the use of IPTG which interferes with our LacI repressor in the system. This prevents an issue as there are few standardized protocols for expressing our protein while minimizing the inclusion bodies or metabolic strains that could impair bacterial growth.

Therefore, for our project, to prevent possible Ago-2 inclusion body formations, we tested various environmental conditions by incubating bacteria at 25°C on a shaker with the speed at 250 rpm for 4 or 6 hours, or overnight. Unfortunately, despite our best efforts, these optimizations did not seem to improve our incubation results, and so we strongly recommend future iGEM teams to test other temperatures and incubation times.

Notes on TEDA

(1). As discussed in the Experiments page, T5-exonuclease dependent assembly (TEDA) was utilized for cloning over the Gibson Assembly. We chose TEDA due to advice from advisors and evidence from our literature search that TEDA was shown to yield a higher cloning efficiency over the traditional Gibson Assembly. Further, TEDA is a much cheaper and accessible alternative compared to Gibson Assembly, as it only requires the use of a single enzyme, a T5-exonuclease, which costs 25 cents (USD) per reaction (Xia et al., 2018). This is much simpler compared to Gibson Assembly, which also uses a T5-exonuclease, but additionally requires 2 additional enzymes, DNA polymerase and ligase, which respectively fill the gaps in DNA and covalently link them. Further, Gibson assembly is conducted in vitro. TEDA solely uses the T5 exonuclease, which chews at the 5’ end, creating overhangs which naturally anneal with each other, removing the need for additional enzymes. Any further gap repair and covalent linkage is done in vivo, by the E.coli cell’s own endogenous machinery. Thus TEDA was used for cloning each of our plasmids in our E.coli strain.

(2). We would like to provide some tips and further information for any future iGEM teams that may be considering TEDA for their cloning. First, we would like to note that assembling base pairs of 100 nucleotides and below was typically unsuccessful for us. All of our successful transformations occurred with fragments that were at least 1000 base pairs. We would recommend cloning genes of that size or larger, although some experimentation could be done with fragments in between the range 100, which we found to be unsuccessful, and a 1000.

(3). Second, one can generally follow primer design instructions for Gibson. We particularly recommend utilizing Snapgene’s primer design function, as we discuss on our Experiments page. This notably does not involve having to accommodate a restriction enzyme site; the nature of the protocol allows it such that your primers will only include your template complementary to the existing DNA and the portion complementary to the fragment you want to add.

(4). To compare the efficiency of TEDA’s transformation component, we compared our positive control for one TEDA transformation utilizing a generic plasmid, PUC19, with plates that the 2023 Stony Brook iGEM Team prepared with the same plasmid. They utilized TSS transformation, which involves the TSS buffer which is distinct from TEDA reaction mixture to modify cells prior to transformation (Pawlowski, 2020), and Inoue, which involves growing cells at 18 degrees Celsius rather than the usual 37 and a different chemical combination (Im et al., 2011).

(5). It can be observed that in this case the heat shock transformation of TEDA was as effective in transforming the plasmids as the methods utilized by the 2023 Stony Brook iGEM Team. Note that the plates for Inoue exhibited overgrowth due to storage for a year, though this did not occur as dramatically in the plates for TSS.

TEDA/heat shock plates from this year, including a test for Ampicillin resistance gene in the PUC19 plasmid.

TSS plates from last year that tested for Ampicillin resistance gene on the PUC19 plasmid.

Inoue plates from last year that tested Ampicillin resistance gene on the PUC 19 plasmid.