Part:BBa_K5490024
REPORTER FOR TESTING WNV GRNA TARGET SITES
It’s a luciferase reporter gene with three 23-base-long sequences that can be targeted by endonuclease enzymes such as RNAi or Cas13. The reporter can be analyzed either through microscopic observation, as it is fused with an unstable EGFP, or through a luciferase assay to evaluate the interaction between the endonuclease and the target sites.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 3271
Illegal XbaI site found at 3493
Illegal PstI site found at 1490 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 3271
Illegal PstI site found at 1490
Illegal NotI site found at 3476 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 3271
Illegal BamHI site found at 3484 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 3271
Illegal XbaI site found at 3493
Illegal PstI site found at 1490 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 3271
Illegal XbaI site found at 3493
Illegal PstI site found at 1490
Illegal NgoMIV site found at 1652
Illegal NgoMIV site found at 3029
Illegal NgoMIV site found at 3050
Illegal NgoMIV site found at 3365
Illegal AgeI site found at 2744 - 1000COMPATIBLE WITH RFC[1000]
Usage and Biology
This composite part consists of two distinct reporter systems designed for effective monitoring of gene activity. Upstream is an unstable eGFP reporter, which is ideal for live-cell imaging, allowing researchers to visualize cellular processes in real time. Downstream, there is a luciferase gene fused with degradation sites to enhance the responsiveness of the signal.
The luciferase component features three target sites, each 23 nucleotides in length, that are found within the WNV genome. Two of these target sites are located upstream of a flexible joint, while the third is situated just upstream of the poly(A) signal. These sequences have been selected using an algorithmic approach, identifying them as optimal targets for the effector molecule CasRx.
One of the advantages of this composite design is the flexibility it offers researchers: they can easily swap out these target sequences for any sequences they wish to target using RNAi or Cas13. The unstable nature of both reporters ensures that the reporter signal will cease almost immediately after cleavage occurs, providing a rapid and clear indication of gene activity.
By employing two reporters, this system enables dual-method validation. Researchers can assess gene expression in real time using live-cell imaging under a microscope, while also conducting quantitative measurements in a luciferase assay post-cleavage. This combination of methodologies enhances the robustness of experimental results, allowing for comprehensive analysis of gene regulation and interference efficiency.
Andersen JB, Sternberg C, Poulsen LK, Bjorn SP, Givskov M, Molin S. New unstable variants of green fluorescent protein for studies of transient gene expression in bacteria. Appl Environ Microbiol. 1998 Jun;64(6):2240-6. doi: 10.1128/AEM.64.6.2240-2246.1998. PMID: 9603842; PMCID: PMC106306.
Huttly A. Reporter genes. Methods Mol Biol. 2009;478:39-69. doi: 10.1007/978-1-59745-379-0_3. PMID: 19009438.
Meyer RD, Srinivasan S, Singh AJ, Mahoney JE, Gharahassanlou KR, Rahimi N. PEST motif serine and tyrosine phosphorylation controls vascular endothelial growth factor receptor 2 stability and downregulation. Mol Cell Biol. 2011 May;31(10):2010-25. doi: 10.1128/MCB.01006-10. Epub 2011 Mar 14. PMID: 21402774; PMCID: PMC3133358.
Conti E, Franks NP, Brick P. Crystal structure of firefly luciferase throws light on a superfamily of adenylate-forming enzymes. Structure. 1996 Mar 15;4(3):287-98. doi: 10.1016/s0969-2126(96)00033-0. PMID: 8805533.
Chng J, Wang T, Nian R, Lau A, Hoi KM, Ho SC, Gagnon P, Bi X, Yang Y. Cleavage efficient 2A peptides for high level monoclonal antibody expression in CHO cells. MAbs. 2015;7(2):403-12. doi: 10.1080/19420862.2015.1008351. PMID: 25621616; PMCID: PMC4622431.
Structural Experiments and Engineering Process
BUILDING THE REPORTER INSIDE PEGFPC1
Designing leads to safety
We ordered an insert containing a degradation signal derived from the pcDNA3.3_d2eGFP construct, a T2A peptide, and the luciferase gene with three target sequences. To facilitate directional cloning, we included two restriction sites: HindIII upstream and BamHI downstream. These allowed us to clone the insert into a plasmid already available in the lab. The plasmid, pEGFPC1, contains a CMV promoter, an EGFP gene, and a multicloning site that includes both the HindIII and BamHI restriction sites, with a poly-A tail downstream.
Building a safe virus
Vector Preparation: Initially, we performed a double digestion on the multi-linker of the vector using HindIII and BamHI. This generated a linearized vector with two distinct sticky ends. Although the vector contained two non-matching sticky ends, we opted to treat the vector with alkaline phosphatase to prevent self-ligation, ensuring a more efficient ligation process with the insert.
Insert Preparation: We similarly performed a double digestion on the insert with HindIII and BamHI, followed by a gel extraction step to obtain an insert with two sticky ends compatible with the vector's ends.
Ligation Step: The insert and the vector were mixed in a 2:1 molar ratio, followed by the addition of T4 ligase. The ligation mixture was then transformed into DH5α cells. Multiple control groups were included: one without T4 ligase, one without alkaline phosphatase, and groups missing either or both of these components. These controls were critical for assessing the ligation efficiency and ensuring the success of the cloning strategy.
All of the above constructs were successfully transformed and amplified in DH5α cell lines.
Testing
Initially, we compared the transformation efficiency between the experimental group and all control groups. Subsequently, we screened various colonies from the experimental Petri dishes. We first lysed the colonies using alkaline lysis, followed by restriction digestion to identify the correct plasmid and eliminate any background colonies.
The digestions where performed with EcoRI that cuts within the insert and HindIII that doesn't cut in the insert that was cloned. If the cloning should be succesful , then we should observe 2 bands , at 5010 and 1800 . On this electrophoresis gel the colonies are placed first as digested and then as supercoiled. As we can tell every digested colony ,has 3 bands because the quantity of enzyme was not enough to cut all of the plasmid ,but we can observe the wanted bands at 5010 and 1800. Agarose Gel 0.8% Marker 1kb Ladder plus by NEB
Through this process, we successfully identified multiple colonies containing the desired plasmid, which were further validated using various restriction digest assays to confirm the presence of the correct inserts.
Learn
Through the design of this reporter, we gained valuable insights into the role of degradation signals that can significantly reduce the half-life of any protein. This knowledge emphasized the importance of careful consideration when fusing degradation signals with EGFP to preserve the open reading frame (ORF). Additionally, we learned about the importance of dephosphorylation of the vector as even when having different sticky ends, this can influence the production of background colonies.
Lastly, despite the emergence of numerous new cloning methods, we found that traditional techniques, such as directional cloning, often remain straightforward and highly effective strategies for producing constructs. This reinforces the idea that sometimes the simplest methods can yield the best results in molecular cloning.
Fuctional Experiments
Experiments Viral Reporter ICC:
Since no fluorescence was observed in the live cell imaging, we performed immunocytochemistry (ICC) with an antibody against EGFP to detect the reporter protein. Cells were transfected with 20% dsRED and 90% p.EGFP-C1-LUC reporter and subsequently observed under a confocal microscope.
Results and Discussion Viral Reporter ICC:
Neuro2a
Unfortunately, we did not observe any signal in either of the cell lines. A possible interpretation for this could be that EGFP is extremely unstable, or that the low luciferase activity indicates the entire construct may be unstable.
Viral Reporter Luciferase Assay:
Experiments in Neuro2a Cell Line
Initially, Neuro2a cells were transfected with the dsRED plasmid and observed under a fluorescence microscope to measure transfection efficiency.
We performed transfections on the cell lines, using the following plasmids as controls to predict luminescence, which could then be compared with our experimental groups. The well containing 10% dsRED and 90% CMV-EGFP-C1 plasmids will not produce any luminescence, as it lacks the necessary luciferase component. This serves as our negative control.
The other wells contain positive controls, arranged in incremental order based on expected luminescence production:
10% dsRED and 90% Basic-LUC vector
10% dsRED and 90% SV40 Enhancer-LUC vector
10% dsRED and 90% SV40 Promoter-LUC vector
10% dsRED and 90% SV40-LUC control vector
10% dsRED and 90% CMV-LUC vector
Note: These types of controls were utilized for both the viral reporter assay and the pRE-LUC assays and in Neuro2a and SHSY-5Y cell lines.
For the experimental group, we transfected the cells with 10% dsRED and 90% CMV-EGFP-C1-reporter, which is our synthesized plasmid.
Results and discussion:
As shown in both the diagram and the table, we observe the expected results across all control groups. There is a clear increase in luminescence from the plasmids anticipated to produce higher amounts of luciferase protein. Normalization was performed using the total negative control 10%DsRED+90% CMV-EGFP-C1.
Finally, in the experimental group, we observed that although luminescence was significantly reduced, the luciferase protein remained active even after the insertion of the three target sequences
Experiments in SHSY-5Y Cell Line Initially, SHSY-5Ycells were transfected with the dsRED plasmid and observed under a fluorescence microscope to measure transfection efficiency.
Note: Pictures are not provided due to the low intensity observed under the microscope.
We performed transfections on the cell lines, using the following plasmids as controls to predict luminescence, which could then be compared with our experimental groups. The well containing 10% dsRED and 90% CMV-EGFP-C1 plasmids will not produce any luminescence, as it lacks the necessary luciferase component. This serves as our negative control.
The other wells contain positive controls, arranged in incremental order based on expected luminescence production:
10% dsRED and 90% Basic-LUC vector
10% dsRED and 90% SV40 Enhancer-LUC vector
10% dsRED and 90% SV40 Promoter-LUC vector
10% dsRED and 90% SV40-LUC control vector
10% dsRED and 90% CMV-LUC vector
Note: These types of controls were utilized for both the viral reporter assay and the pRE-LUC assays and in Neuro2a and SHSY-5Y cell lines.
For the experimental group, we transfected the cells with 10% dsRED and 90% CMV-EGFP-C1-reporter, which is our synthesized plasmid.
Results and discussion:
As shown in both the diagram and the table, we observe the expected results across all control groups. There is a clear increase in luminescence from the plasmids anticipated to produce higher amounts of luciferase protein. Normalization was performed using the total negative control 10%DsRED+90% CMV-EGFP-C1.
Unfortunately, in this cell line, we observed no luminescence from the experimental group, as the luminescent intensity was indistinguishable from that of the negative control group, as shown in the diagram and table.
Neuro2a
When comparing the control groups in the two cell lines we observe that SHSY-5Y cells produce at least 10 times lower levels of luciferase than N2A cells which leads to lower values RLUs/ug of total protein.
Neuro2a
When comparing the experimental groups in the two cell lines, we observed that the light intensity from Neuro2a was small but detectable, whereas in SHSY-5Y, it was essentially non-existent.
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