Difference between revisions of "Part:BBa K1614000"

(iGEM Marburg 2021 - Contribution)
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===iGEM Marburg 2021 - Contribution===
 
===iGEM Marburg 2021 - Contribution===
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A significant difference in expression in cell-free systems is caused by the addition of different DNA concentrations[https://doi.org/10.1021/acssynbio.9b00437<nowiki>[1]</nowiki>].  
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Despite many advantages and simplicity of the system, significant differences in expression using cell-free systems can be caused by the addition of different amounts of template DNA[https://doi.org/10.1021/acssynbio.9b00437<nowiki>[1]</nowiki>]. For this reason, we decided to test the behavior of our system to different DNA concentrations.
Therefore we wanted to test the behavior of our system towards different DNA concentrations.  
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In the following experiment,  NanoLuc luciferase was used as a reporter system, within our cell-free expression measurements. The graph below indirectly shows the expression levels of this NanoLuc luciferase via the emitted luminescence (see Figure 1). To analyse the influence of DNA concentration to the target protein expression level, extracts from two different plant species (N. tabacum and S. oleracea) have been used and DNA concentrations in the spectrum from 0.5 nM to 15 nM have been added to the final cell-free reaction mix. <br>
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For both N. tabacum and S. oleracea, an optimum expression level is reached at  DNA concentration of 5 nM. With a further increase of the concentration, no significant differences in expression levels can be recognised.<br>
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These results show that it is essential to carefully normalize DNA concentration for the comparison of various parts in the experiments using cell-free systems. Additionally, using saturated DNA concentrations has the advantage of being less prone to variations in expression, caused by the use of different concentrations in DNA.
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Moreover it should be “good practice” not to have DNA concentrations as a limiting factor in your measurements, as this could cover other effects/properties one would like to actually characterize in the experiment.
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[[Image:T--Marburg--DNA titration T7 universal test construct.png|thumb|left|800px|<u>Figure 4:</u> <b>Differences in expression of the Nanoluc luciferase in response to the amount of template DNA added to the cell-free reaction</b> Luminescence values are given as arbitrary units and the data is presented on a logarithmic scale. The reaction was set up with a total volume of 10µl and reaction mixtures were set up by creating a premix with all the components except the DNA template.
 
Negative controls using only the plasmid DNA or the crude chloroplast extracts have been included respectively.
 
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[[Image:T--Marburg--DNA titration T7 universal test construct.png|thumb|left|800px|<u>Figure 4:</u> <b>Differences in expression of the NanoLuc luciferase in response to the amount of template DNA added to the cell-free reaction</b> Luminescence values are given as arbitrary units and the data is presented on a logarithmic scale. The reaction was set up with a total volume of 10µl and reaction mixtures were set up by creating a premix with all the components except DNA template.
 +
Negative controls using only the plasmid DNA or the crude chloroplast extracts have been included respectively
  
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]]
  
<p>
 
In the following experiment, the Nanoluc luciferase was used as a reporter system, within our cell-free expression measurement. The graph indirectly shows the expression levels of this Nanoluc luciferase via the emitted luminescence. Extracts from two different species have been utilized and DNA concentrations in the range from 0.5nM to 15nM have been added to the final cell-free reaction to analyse the effect of the DNA concentration on the expression level.
 
For both <i>N. tabacum</i> and <i>S. oleracea</i>, an optimum is reached at a DNA concentration of 5nM. With a further increase of the concentration, no significant difference in the luminescence can be detected.
 
<br><br>
 
These results show that it is of great importance for experiments using cell-free systems to carefully normalize the DNA concentration for the comparison of a variety of parts.
 
Additionally, using saturated DNA concentrations has the advantage of being less prone to variations in expression, caused by the use of different DNA concentrations.
 
Moreover, it should be “good practice” to not have DNA concentrations as a limiting factor in your measurements, as this could conceal other effects/properties one would like to actually characterize in the experiment.
 
</p>
 
 
 
</html>
 
  
 
==References==
 
==References==
  
 
[1] Kopniczky, M. B., Canavan, C., McClymont, D. W., Crone, M. A., Suckling, L., Goetzmann, B., Siciliano, V., MacDonald, J. T., Jensen, K., & Freemont, P. S. (2020). Cell-Free Protein Synthesis as a Prototyping Platform for Mammalian Synthetic Biology. In ACS Synthetic Biology (Vol. 9, Issue 1, pp. 144–156). American Chemical Society (ACS). https://doi.org/10.1021/acssynbio.9b00437
 
[1] Kopniczky, M. B., Canavan, C., McClymont, D. W., Crone, M. A., Suckling, L., Goetzmann, B., Siciliano, V., MacDonald, J. T., Jensen, K., & Freemont, P. S. (2020). Cell-Free Protein Synthesis as a Prototyping Platform for Mammalian Synthetic Biology. In ACS Synthetic Biology (Vol. 9, Issue 1, pp. 144–156). American Chemical Society (ACS). https://doi.org/10.1021/acssynbio.9b00437

Revision as of 00:57, 20 October 2021

T7 promoter for expression of functional RNA

Minimal promoter derived from T7 phage promoter.


Usage and Biology

This T7 promoter derivate is especially useful for the use in in vitro applications. The G which is the startpoint for transcription is included in the promoter sequence.

Characterize the T7 promoter – CCU-Taiwan

In our pHMT-LbCas12a construct, we included the T7 promoter (Part:BBa_K1614000) to induce the LbCas12a expression. To characterize the ability of T7 promoter to induce functional RNA, we examined whether the T7 promoter induced LbCas12a RNA can be translated into proteins by SDS-PAGE and Coomassie blue staining. We first transformed pHMT-LbCas12a into E.coli BL21(DE3), which harbors a Lac Operon regulated T7 polymerase. We then expanded the transformed BL21(DE3) cells by TB medium at 37℃. When O.D.600 approach 0.6 ~ 0.8, we induced T7 polymerase expression by adding 0.2 mM IPTG to TB medium and shift BL21(DE3) to 16℃ for 16 hr.

The IPTG treatment increased the T7 polymerase expression in BL21(DE3) as time goes on, which in turn activate T7 promoter. The activated T7 promoter should promote LbCas12 transcription and translation. To examine this, we collected IPTG-induced BL21(DE3) every two hour, and performed SDS-PAGE assay and Coomassie Blue staining to detect LbCas12a protein expression. In brief, 1 ml of BL21(DE3) is harvested every two hour and centrifuged to cell pellet. The cell pellet was then sonicated, and soluble fraction in supernatant was collected by centrifuging at 13200 rpm 30 min. Total 1 ml supernatant, which equal to protein expression in 1ml BL21(DE3), was used in SDS-PAGE and Coomassie Blue staining. To calculate the protein expression at different time points, we quantity the image intensity of LbCas12a protein and BSA standard by Image J.

  1. Coomassie blue staining shows the LbCas12a protein expression was absent at 0 hr and increased as time goes on. This result indicated that T7 promoter can be activated by IPTG-induced T7 polymerase (Figure 1).
  2. We then used BSA standard to quantify the LbCas12a protein expression. The simple linear regression of BSA protein expression standard is shown in Figure 2, while the original image intensity detected by Image J is shown in table 1.
  3. Finally, we quantify the LbCas12 expression by conversing the intensity to concentration through BSA standard regression (Figure 3).
Figure 1: The Coomassie Blue staining of protein expression iPTG-induced BL21(ED3) at different time points and the BSA protein standard. The total protein expression in IPTG induced BL21(DE3) at 4, 8, 10 ,12, 14, and 16 hr were shown. The 35, 70, 210, 350 ng BSA was loaded as standard.
Figure 2: The simple linear regression of BSA protein standard.The 35, 70, 210, 350 ng BSA was used as protein standard to perform simple linear regression with image intensity.
Figure 3: The quantification of LbCas12 protein expression at different time points of ITGP induction.


Table 1: Image quantification BSA standard of Figure 1
BSA (ng) Background intensity BSA intensity BSA - Background intensity
350 2407241 1258862 1148379
210 1589271 962468 626803
70 1544912 904623 180999
35 1042670 861671 129877


Table 1: Table 2: Image quantification LbCas12a expression of Figure 1
Time (hours) Background intensity LbCas12a intensity LbCas12a - Background intensity LbCas12a (ng) LbCas12a (µg/mL)
0hr +IPTG 696645 691258 5387 8.937 2.554
4hr +IPTG 900536 760105 140431 50.098 14.314
8hr +IPTG 925618 789444 136174 48.801 13.943
10hr +IPTG 913959 791540 122419 44.608 12.745
12hr +IPTG 956220 770282 185938 63.968 18.277
14hr +IPTG 976568 761481 215087 72.853 20.815
16hr +IPTG 1064337 764543 299794 98.671 28.192
16hr Ctrl 924162 743361 180801 62.403 17.829


Conclusion: The quantification of LbCas12a protein expression by BSA standard shows that the Cas12 protein is increased at different time points, which confirmed that the T7 promoter is functional in our construct.


iGEM Marburg 2021 - Contribution

Cell-free transcription and translation systems are advanced in vitro used as prototyping platforms for metabolic networks and gene circuits. Typically, they are based on crude cell extracts and contain the whole machinery needed for protein biosynthesis. This advantage in cell-free systems offers exciting opportunities to fundamentally transform synthetic biology. It enables new approaches for model-driven design of synthetic gene networks, rapid and portable acquisition of targeted components, as well as on-demand biomanufacturing, and building synthetic cells from scratch. Recently, cell-free technology approaches have gained more and more importance in the synthetic biology field. While the majority of cell-free systems are based on bacterial and eukaryotic cells, there are almost no cell-free systems based on plants. For this reason, our project was focused on developing novel cell-free systems of chloroplasts from various plant species. With our Chloroplast Cell-Free system we provide a unique test-platform, which can be easily applied to study various genetic constructs for plant engineering in a much shorter time frame.



The DNA concentration response of the T7 polymerase in a chloroplast cell-free system

Despite many advantages and simplicity of the system, significant differences in expression using cell-free systems can be caused by the addition of different amounts of template DNA[1]. For this reason, we decided to test the behavior of our system to different DNA concentrations.

In the following experiment, NanoLuc luciferase was used as a reporter system, within our cell-free expression measurements. The graph below indirectly shows the expression levels of this NanoLuc luciferase via the emitted luminescence (see Figure 1). To analyse the influence of DNA concentration to the target protein expression level, extracts from two different plant species (N. tabacum and S. oleracea) have been used and DNA concentrations in the spectrum from 0.5 nM to 15 nM have been added to the final cell-free reaction mix.
For both N. tabacum and S. oleracea, an optimum expression level is reached at DNA concentration of 5 nM. With a further increase of the concentration, no significant differences in expression levels can be recognised.
These results show that it is essential to carefully normalize DNA concentration for the comparison of various parts in the experiments using cell-free systems. Additionally, using saturated DNA concentrations has the advantage of being less prone to variations in expression, caused by the use of different concentrations in DNA. Moreover it should be “good practice” not to have DNA concentrations as a limiting factor in your measurements, as this could cover other effects/properties one would like to actually characterize in the experiment.


Figure 4: Differences in expression of the NanoLuc luciferase in response to the amount of template DNA added to the cell-free reaction Luminescence values are given as arbitrary units and the data is presented on a logarithmic scale. The reaction was set up with a total volume of 10µl and reaction mixtures were set up by creating a premix with all the components except DNA template. Negative controls using only the plasmid DNA or the crude chloroplast extracts have been included respectively


References

[1] Kopniczky, M. B., Canavan, C., McClymont, D. W., Crone, M. A., Suckling, L., Goetzmann, B., Siciliano, V., MacDonald, J. T., Jensen, K., & Freemont, P. S. (2020). Cell-Free Protein Synthesis as a Prototyping Platform for Mammalian Synthetic Biology. In ACS Synthetic Biology (Vol. 9, Issue 1, pp. 144–156). American Chemical Society (ACS). https://doi.org/10.1021/acssynbio.9b00437