Difference between revisions of "Part:BBa K2807012"

Latest revision as of 09:33, 17 October 2018

eGFP-T2A-mCherry

Usage and Biology

Inspired by the work of Martin et al. (2018), as well as WPI Worcester 2016 iGEM project, we designed a eGFP-T2A-mCherry dual fluorescence reporter plasmid system. This plasmid consists of two fluorescence markers, an EGFP gene and a mCherry gene. Here, mCherry is expressed constitutively and used as a marker for transfection efficiency and expression levels of a base editing fusion protein, dCAS-XTEN-APOBEC (BBa_K2807003).

Characterisation

1. Mutation of the start codon, ATG to ACG in EGFP can significantly block the EGFP expression without affecting expression of mCherry

To demonstrate that a single nucleotide mutation in the start codon could result in ON to OFF change in EGFP expression in cells, we cloned both the EGFP-T2A-mCherry-WT (BBa_K2807012) and the EGFP-T2A-mCherry-ACG mutant (BBa_K2807013) into C1 mammalian expression vector for expression in HEK293T cells. We transfected HEK293T cells with both wild type (WT) reporter and ACG mutated (ACG) reporter and thereafter, imaged them via microscopy and harvested them for flow cytometry analysis.

As shown in Figure 1, the cells transfected with EGFP-T2A-mCherry-WT reporter expressed both GFP and mCherry proteins in the same cells, showing that T2A self cleavage peptide is effective. We then further quantified the level of fluorescence by flow cytometry and our results showed that at least 36.6% of the transfected cells are both EGFP+/mCherry+ (Figure 3). There are about 7.9% of the transfected cells with EGFP signal only. This is expected because it is possible for the ribosome to fall off from the mRNA when it encounters the T2A signal peptide and cannot continue with translation. As a result, only EGFP protein is produced but not mCherry.

On the other hand, HEK293T cells transfected with EGFP-T2A-mCherry-ACG mutant showed red fluorescence expression, with very low or undetectable green fluorescent protein levels as visualised via microscopy (Figure 2). Our experiment showed that the mutation of the start codon in EGFP is indeed effective in abolishing the expression of EGFP without affecting the expression of the mCherry protein. Compared to the cells transfected with WT reporter, the percentage of EGFP+/mCherry+ positive cells in cells transfected with the EGFP-T2A-mCherry-ACG mutant is almost undetectable (Figure 3). In addition, the mean fluorescence intensity for mCherry in cells transfected with EGFP-T2A-mCherry-WT and cells transfected with EGFP-T2A-mCherry-ACG mutant are similar, while there is significant reduction in EGFP expression between the two groups of cells.

Figure 1. Representative fluorescent images of ACG mutant reporter (ACG) wild type reporter (WT) transfected in HEK293T cells. Images taken at 60X magnification, scale bar = 50um.

Figure 2. Representative fluorescent images of ACG mutant reporter (ACG) wild type reporter (WT) transfected in HEK293T cells. Images taken at 10X magnification, scale bar = 400um.

Figure 3. Flow cytometry analysis of EGFP and mCherry expression on wild type (WT) and ACG mutant (ACG) reporters transfected in HEK293T cells. (A) Strong correlation between expression level of EGFP and mCherry. (B) No leaky expression of EGFP in mutant ACG reporter. (C) Percentage of EGFP mCherry double positive cells. (D): Quantification on mean fluorescent intensity. Error bar represents SEM, n=3.

2. The EGFP-T2A-mCherry bicistronic reporter is a significant improvement over the reporter system from a previous iGEM team.

An important feature of a reliable reporter system is to have an internal control signal such that the reporter signal can be normalized to account for stochastic processes in cells. Expression of both EGFP and mCherry reporter on separate plasmids may lead to uneven gene expression due to stochastic processes. hus, it is more advantageous to have both EGFP and mCherry on the same plasmid. Therefore, we designed our reporter system with this intention. We then carried out experiments to compare our reporter system with the reporter system described by the Worcester 2016 team (http://2016.igem.org/Team:WPI_Worcester). We evaluated the system in terms of background noise, as well as the reliability of the internal control.

We performed double transfection of the mCherry plasmid with either ATG EGFP (BBa_K2807009) or ACG EGFP (BBa_K2807010) plasmid in HEK293T cells. As shown in Figure 4, WPI reporters showed the expected OFF to ON change from ACG mutant to ATG. However, the fluorescence intensity of EGFP and mCherry does not correlate well with one another, and there was some leaky expression of EGFP even in the mutant form (Figure 5A). Moreover, 2.3% of the cells are double positive for EGFP in cells expressing the WPI ACG mutant reporter (Figure 5B & 5C), which is higher than our ACG bicistronic reporter construct (Figure 3B).

On the other hand, our bicistronic reporter construct showed a strong linear correlation between EGFP and mCherry fluorescence intensity in WT reporter (Figure 2A), and the number of cells positive for EGFP in our ACG mutant construct is effectively non-detectable. Therefore, our dual reporter system provides higher signal to noise ratio and allows for the quantification of relative editing efficiency between different cells and in different transfection experiments.

Figure 4. Fluorescence imaging of WPI 2016 EGPF reporters in transfected cells. HEK293T cells in a 6-well plate are transfected with 1ug of EGFP and mCherry plasmids each, and photos were taken 24 hrs post transfection. (A) EGFP expression in wild type ATG EGFP transfected cells. (B) mCherry expression when co-transfected with EGFP. (C) EGFP expression in mutant ACG EGFP transfected cells. There are a few cells with weak EGFP expression. (D) mCherry expression when co-transfected with EGFP.

Figure 5. Flow cytometry analysis on EGFP and mCherry expression on Worcester 2016’s wild type and ACG mutant reporters. HEK293T cells were co-transfected with wild type or mutant EGFP. (A) No clear correlation was observed between expression level of EGFP and mCherry. (B) Leaky expression of EGFP is observed in mutant ACG reporter. (C) Percentage of EGFP mCherry double positive cells. (D): Quantification on mean fluorescent intensity. Error bar represents SEM, n=3.

3. EGFP-T2A-mCherry Reporter can report DNA editing efficiency of Base Editors

Although the EGFP-T2A-mCherry Reporter was initially designed for RNA editing, we reasoned that this construct could also be used to report Cas9 editing efficiency in DNA strands. Base editor 3 and 4 (BE3, BE4) (Komer et al., 2016, Komer et al., 2017), fused with the nuclease-deficient dCas9n to the enzyme APOBEC can deaminate C to U in DNA templates, which restores the DNA sequence to T after replication.

As such, we tested out our reporter system using BE3 and BE4 plasmids. HEK293T cells were transfected with base editor, ACG reporter and gRNA concurrently and the number of DNA editing events was quantified using flow cytometry. We observed that among the mCherry expressing cells, 60% of them have GFP fluorescence as a result of base correction from ACG to ATG (Figure 6,7). The EGFP fluorescence intensity was also restored to a level comparable to the wild type reporter (Table 1), confirming the occurrence of editing events. On the other hand, transfecting cells with only the base editors and ACG reporter showed no EGFP fluorescence, indicating that the editing event is specific and directed by the gRNA sequence.

Figure 6. Fluorescent imaging of cells after DNA base editing. HEK293T cells were co-transfected with base editor 3 (BE3), gRNA plasmid, and ACG reporter (A,B), or base editor 4 (BE4), gRNA plasmid, and ACG reporter (C,D), or Base editor 3 (BE3) and ACG reporter (E,F). Images were taken 16 hours post transfection. A-D: 10X magnification, scale bar =400um. E-F: 40X magnification, scale bar =100um.

Figure 7. Comparison of editing efficiency using BE3 and BE4. HEK293T cells were transfected with ACG eGFP-T2A-mCherry reporter with (A) BE3 and gRNA, (B) BE4 and gRNA, (C) BE3 only as control.

**Table 1. Quantification of base editors (BE3,BE4) editing efficiency using eGFP-T2A-mCherry reporter. Cells are co-transfected with ACG reporter and indicated plasmid.**
Sample	EGFP Mean Fluorescent intensity	mCherry Mean Fluorescent Intensity	Percentage of EGFP+ cells among mCherry+ cells
BE3+gRNA	5909	6286	60.6%
BE4+gRNA	5671	6067	62.3%
BE3	816	5456	0.4%

Conclusion

In conclusion, our EGFP-T2A-mCherry reporter system is able to achieve a clear OFF-to-ON switch upon base editing from C to U. It improved the part from the previous iGEM team project by adding mCherry (as transfection control) in the same reporter plasmid. In cells expressing our constructs, the expression efficiency, EGFP and mCherry expression showed strong correlation with transfection rates. In addition, our reporter has been shown to work for Cas9-mediated DNA editing, thus providing an alternative tool for real time, in vitro visualization and reporting of base editing efficiency.

References

Komor, A. C., Kim, Y. B., Packer, M. S., Zuris, J. A., & Liu, D. R. (2016). Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature, 533(7603), 420.

Komor, A. C., Zhao, K. T., Packer, M. S., Gaudelli, N. M., Waterbury, A. L., Koblan, L. W., ... & Liu, D. R. (2017). Improved base excision repair inhibition and bacteriophage Mu Gam protein yields C: G-to-T: A base editors with higher efficiency and product purity. Science advances, 3(8), eaao4774.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 470
Illegal NheI site found at 493
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 126
1000
COMPATIBLE WITH RFC[1000]

@@ Line 16: / Line 16: @@
 On the other hand, HEK293T cells transfected with EGFP-T2A-mCherry-ACG mutant showed red fluorescence expression, with very low or undetectable green fluorescent protein levels as visualised via microscopy (Figure 2). Our experiment showed that the mutation of the start codon in EGFP is indeed effective in abolishing  the expression of EGFP without affecting the expression of the mCherry protein. Compared to the cells transfected with WT reporter, the percentage of EGFP+/mCherry+ positive cells in cells transfected with the EGFP-T2A-mCherry-ACG mutant is almost undetectable  (Figure 3). In addition, the mean fluorescence intensity for mCherry in cells transfected with EGFP-T2A-mCherry-WT and cells transfected with EGFP-T2A-mCherry-ACG mutant are similar, while there is significant reduction in EGFP expression between the two groups of cells.
-https://static.igem.org/mediawiki/2018/a/a3/T--NUS_Singapore-Sci--dr_fig1.png
+[[Image:DR Fig1.png|800px|center|]]
-'''Figure 1. Representative fluorescent images of ACG mutant reporter (ACG) wild type reporter (WT) transfected in HEK293T cells.''' Images taken at 60X magnification, scale bar = 50um.
+'''Figure 1. Representative fluorescent images of ACG mutant reporter (ACG) wild type reporter (WT) transfected in HEK293T cells.''' ''Images taken at 60X magnification, scale bar = 50um.''
-https://static.igem.org/mediawiki/2018/f/ff/T--NUS_Singapore-Sci--dr_fig2.png
-'''Figure 2. Representative fluorescent images of ACG mutant reporter (ACG) wild type reporter (WT) transfected in HEK293T cells.''' Images taken at 10X magnification, scale bar = 400um.
-https://static.igem.org/mediawiki/2018/a/ac/T--NUS_Singapore-Sci--dr_fig3.png
+[[Image:DR Fig2.png|800px|center|]]
-'''Figure 3. Flow cytometry analysis of EGFP and mCherry expression on wild type (WT) and ACG mutant (ACG) reporters transfected in HEK293T cells.''' (A) Strong correlation between expression level of EGFP and mCherry. (B) No leaky expression of EGFP in mutant ACG reporter. (C) Percentage of EGFP mCherry double positive cells. (D): Quantification on mean fluorescent intensity. Error bar represents SEM, n=3.
+'''Figure 2. Representative fluorescent images of ACG mutant reporter (ACG) wild type reporter (WT) transfected in HEK293T cells.''' ''Images taken at 10X magnification, scale bar = 400um.''
+[[Image:DR Fig3.png|800px|center|]]
+'''Figure 3. Flow cytometry analysis of EGFP and mCherry expression on wild type (WT) and ACG mutant (ACG) reporters transfected in HEK293T cells.''' ''(A) Strong correlation between expression level of EGFP and mCherry. (B) No leaky expression of EGFP in mutant ACG reporter. (C) Percentage of EGFP mCherry double positive cells. (D): Quantification on mean fluorescent intensity. Error bar represents SEM, n=3.''
 <h2><span lang="EN-US">2. The EGFP-T2A-mCherry bicistronic reporter is a significant improvement over the reporter system from a previous iGEM team.</span></h2>
@@ Line 29: / Line 32: @@
 An important feature of a reliable reporter system is to have an internal control signal such that the reporter signal can be normalized to account for stochastic processes in cells. Expression of both EGFP and mCherry reporter on separate plasmids may lead to uneven gene expression due to stochastic processes. hus, it is more advantageous to have both EGFP and mCherry on the same plasmid. Therefore, we designed our reporter system with this intention. We then carried out experiments to compare our reporter system with the reporter system described by the Worcester 2016 team (http://2016.igem.org/Team:WPI_Worcester). We evaluated the system in terms of background noise, as well as the reliability of the internal control.
-We performed double transfection of the mCherry plasmid with either ATG EGFP (<partinfo>BBa_K2807009</partinfo>) or ACG EGFP (<partinfo>BBa_K2807010</partinfo>) plasmid in HEK293T cells. As shown in Figure 4, WPI reporters showed the expected OFF to ON change from ACG mutant to ATG. However, the fluorescence intensity of EGFP and mCherry does not correlate well with one another, and there were some leaky expression of EGFP even in the mutant form (Figure 5A). Moreover, 2.3% of the cells are double positive for EGFP in cells expressing the WPI ACG mutant reporter (Figure 5B & 5C), which is higher than our ACG bicistronic reporter construct (Figure 3B).
+We performed double transfection of the mCherry plasmid with either ATG EGFP (<partinfo>BBa_K2807009</partinfo>) or ACG EGFP (<partinfo>BBa_K2807010</partinfo>) plasmid in HEK293T cells. As shown in Figure 4, WPI reporters showed the expected OFF to ON change from ACG mutant to ATG. However, the fluorescence intensity of EGFP and mCherry does not correlate well with one another, and there was some leaky expression of EGFP even in the mutant form (Figure 5A). Moreover, 2.3% of the cells are double positive for EGFP in cells expressing the WPI ACG mutant reporter (Figure 5B & 5C), which is higher than our ACG bicistronic reporter construct (Figure 3B).
 On the other hand, our bicistronic reporter construct showed a strong linear correlation between EGFP and mCherry fluorescence intensity in WT reporter (Figure 2A), and the number of cells positive for EGFP in our ACG mutant construct is effectively non-detectable. Therefore, our dual reporter system provides higher signal to noise ratio and allows for the quantification of relative editing efficiency between different cells  and in different transfection experiments.
-https://static.igem.org/mediawiki/2018/8/82/T--NUS_Singapore-Sci--dr_fig4.png
+[[Image:DR Fig4.png|800px|center|]]
-'''Figure 4. Fluorescence imaging of WPI 2016 EGPF reporters in transfected cells.''' HEK293T cells in a 6-well plate are transfected with 1ug of EGFP and mCherry plasmids each, and photos were taken 24 hrs post transfection. (A) EGFP expression in wild type ATG EGFP transfected cells. (B) mCherry expression when co-transfected with EGFP. (C) EGFP expression in mutant ACG EGFP transfected cells. There are a few cells with weak EGFP expression. (D) mCherry expression when co-transfected with EGFP.
+'''Figure 4. Fluorescence imaging of WPI 2016 EGPF reporters in transfected cells.''' ''HEK293T cells in a 6-well plate are transfected with 1ug of EGFP and mCherry plasmids each, and photos were taken 24 hrs post transfection. (A) EGFP expression in wild type ATG EGFP transfected cells. (B) mCherry expression when co-transfected with EGFP. (C) EGFP expression in mutant ACG EGFP transfected cells. There are a few cells with weak EGFP expression. (D) mCherry expression when co-transfected with EGFP.''
+[[Image:DR Fig5.png|800px|center|]]
+'''Figure 5. Flow cytometry analysis on EGFP and mCherry expression on Worcester 2016’s wild type and ACG mutant reporters.''' ''HEK293T cells were co-transfected with wild type or mutant EGFP. (A) No clear correlation was observed between expression level of EGFP and mCherry. (B) Leaky expression of EGFP is observed in mutant ACG reporter. (C) Percentage of EGFP mCherry double positive cells. (D): Quantification on mean fluorescent intensity. Error bar represents SEM, n=3.''
-https://static.igem.org/mediawiki/2018/b/bc/T--NUS_Singapore-Sci--dr_fig5.png
-'''Figure 5. Flow cytometry analysis on EGFP and mCherry expression on Worcester 2016’s wild type and ACG mutant reporters.''' HEK293T cells were co-transfected with wild type or mutant EGFP. (A) No clear correlation was observed between expression level of EGFP and mCherry. (B) Leaky expression of EGFP is observed in mutant ACG reporter. (C) Percentage of EGFP mCherry double positive cells. (D): Quantification on mean fluorescent intensity. Error bar represents SEM, n=3.
 <h2><span lang="EN-US">3. EGFP-T2A-mCherry Reporter can report DNA editing efficiency of Base Editors</span></h2>
-Although the EGFP-T2A-mCherry Reporter was initially designed for RNA editing, we reasoned that this construct could also be used to report Cas9 editing efficiency in DNA strands. Base editor 3 and 4 (BE3, BE4) (Komer et al., 2016, Komer et al., 2017), fused with the nuclease-deficient dCas9n to the enzyme APOBEC can deaminate C to U in DNA templates, which restores the DNA sequence to T after replication (ref).
+Although the EGFP-T2A-mCherry Reporter was initially designed for RNA editing, we reasoned that this construct could also be used to report Cas9 editing efficiency in DNA strands. Base editor 3 and 4 (BE3, BE4) (Komer et al., 2016, Komer et al., 2017), fused with the nuclease-deficient dCas9n to the enzyme APOBEC can deaminate C to U in DNA templates, which restores the DNA sequence to T after replication.
 As such, we tested out our reporter system using BE3 and BE4 plasmids. HEK293T cells were transfected with base editor, ACG reporter and gRNA concurrently and the number of DNA editing events was quantified using flow cytometry. We observed that among the mCherry expressing cells, 60% of them have GFP fluorescence as a result of base correction from ACG to ATG (Figure 6,7). The EGFP fluorescence intensity was also restored to a level comparable to the wild type reporter (Table 1), confirming the occurrence of editing events. On the other hand, transfecting cells with only the base editors and ACG reporter showed no EGFP fluorescence, indicating that the editing event is specific and directed by the gRNA sequence.
-https://static.igem.org/mediawiki/2018/7/7b/T--NUS_Singapore-Sci--dr_fig6.png
-'''Figure 6. Fluorescent imaging of cells after DNA base editing.''' HEK293T cells were co-transfected with base editor 3 (BE3), gRNA plasmid, and ACG reporter (A,B), or base editor 4 (BE4), gRNA plasmid, and ACG reporter (C,D), or Base editor 3 (BE3) and ACG reporter (E,F). Images were taken 16 hours post transfection. A-D: 10X magnification, scale bar =400um. E-F: 40X magnification, scale bar =100um.
-https://static.igem.org/mediawiki/2018/0/09/T--NUS_Singapore-Sci--dr_fig7.png
+[[Image:DR Fig6.png|800px|center|]]
-'''Figure 7. Comparison of editing efficiency using BE3 and BE4.''' HEK293T cells were transfected with  ACG eGFP-T2A-mCherry reporter with (A) BE3 and gRNA, (B) BE4 and gRNA, (C) BE3 only as control.
+'''Figure 6. Fluorescent imaging of cells after DNA base editing.''' ''HEK293T cells were co-transfected with base editor 3 (BE3), gRNA plasmid, and ACG reporter (A,B), or base editor 4 (BE4), gRNA plasmid, and ACG reporter (C,D), or Base editor 3 (BE3) and ACG reporter (E,F). Images were taken 16 hours post transfection. A-D: 10X magnification, scale bar =400um. E-F: 40X magnification, scale bar =100um.''
+[[Image:DR Fig7.png|800px|center|]]
+'''Figure 7. Comparison of editing efficiency using BE3 and BE4.''' ''HEK293T cells were transfected with  ACG eGFP-T2A-mCherry reporter with (A) BE3 and gRNA, (B) BE4 and gRNA, (C) BE3 only as control.''
@@ Line 79: / Line 86: @@
    </tr>
 </table>
 '''Conclusion'''
-In conclusion, our EGFP-T2A-mCherry reporter system is able to achieve a clear OFF-to-ON switch upon base editing from C to U. It improved the part from the previous iGEM team project by adding mCherry (as transfection control) in the same reporter plasmid. In cells expressing our constructs, the expression efficiency, EGFP and mCherry expression showed strong correlation with transfection rates. In addition, our reporter has been shown to work for Cas9-mediated DNA editing, thus providing an alternative tool for real time, in vitro visualization and reporting of base editing efficiency.
+In conclusion, our EGFP-T2A-mCherry reporter system is able to achieve '''a clear OFF-to-ON switch''' upon base editing from C to U. It improved the part from the previous iGEM team project by adding mCherry (as transfection control) in the same reporter plasmid. In cells expressing our constructs, the expression efficiency, EGFP and mCherry expression showed strong correlation with transfection rates. In addition, our reporter has been shown to work for Cas9-mediated DNA editing, thus providing an alternative tool for real time, in vitro visualization and reporting of base editing efficiency.