Team:Linkoping Sweden/Demonstrate
Demonstrate
All work done on BBa_K2671420
Plasmids used during the characterization of our biobrick
For simplicity our biobrick BBa_K2671420 is called GroES in the rest of the text. The pGroE7 plasmid codes for both GroES and GroEL, and this system is often called GroE. As seen in fig 1. we had to ligate our biobrick into pSB4A5 to avoid having the same origin of replication and the same antibiotic resistance as any of the other ones we planned to use. We also choose the tetracycline promotor to be able to tune the expression of our biobrick.
Figure 1. Plasmids used in our experiments. pSub. can be either of mNG-Aß1-42, EGFP-Aß1-42, a-synuclein-EGFP and Tau0N4R-EGFP. As seen in the next figure, four combinations were introduced into E.coli (BL21). Substrate, substrate and our biobrick, substrate and our biobrick and GroE, substrate and GroE.
Overview of our experimental design to archive the results shown below
A simple explaination on how we archived our experimental results is shown in figure 2.
Figure 2. The plasmids shown is the same as before, where A can be either of mNG-Aß1-42, EGFP-Aß1-42, a-synuclein-EGFP and Tau0N4R-EGFP. B is always pGroE7 and C is pSB4A5-GroES. The concentrations used for inducing the chaperone plasmids was 0.25 mg/ml L-arabinose, 200 ng/ml tetracycline. For the substrate plasmids we used 0.5 mM IPTG, and the substrate was induced 30 mins after the chaperone plasmids.
Results from the experimental measurements is shown below. The y-axis represent the fluorescence intensity at 520 nm. The excitation was done at 485 nm. The x-axis show the time for the measurements. The second set of graphs shown for each substrate represent the normalized values for the fluorescence intensity. This was analyzed because it represent the kinetic growth of the substrate proteins. As seen in the those graphs, the 50 % of max has been marked out as a dotted line. This makes it easy to interpret if the folding of the different substrate proteins was affected by the chaperone systems.
Results for the mNG-Aß1-42 substrate protein
As seen in the top three graphs our biobrick increases the intensity of the mNG-Aß1-42 protein. However a interesting notice is that in concert with the pGroE7 plasmid, it seem to slow the folding rate significantly as seen in the bottom graphs. A high concentration of GroES, with or without GroEL seems to be the best fit for this substrate protein. While a double-expressed GroES gene in concert with GroEL seems to hinder the production of the substrate more than helping it. This might be because mNG-Aß1-42 is fairly good at folding itself, and a very high level of co-expression, while also dealing with three antibiotics.
Figure 3. Results for mNG-Aß1-42. Top graphs show fluorescene intensity divided by the start OD600 over 16 hours at 37 degrees celsius. The bottom graphs show the normalized values from the top graphs.
Results for the EGFP-Aß1-42 substrate protein
The results for the EGFP-Aß1-42 protein looks quite different compared to the mNG-Aß1-42 protein. In this case our biobrick and the other two combinations show an increase in fluorescence intensity compared to the substrate alone. A interesting notice from the bottom three graphs is that our biobrick without the presence of GroEL slows down the folding rate significantly, and a decrease in intensity is not shown after 16 hours. Perhaps the EGFP-Aß1-42 protein folds better and stays correctly folded better with a low concentration of GroEL.
Figure 4. Results for EGFP-Aß1-42. Top graphs show fluorescene intensity divided by the start OD600 over 16 hours at 37 degrees celsius. The bottom graphs show the normalized values from the top graphs.
Results for the a-synuclein-EGFP substrate protein
The results for the a-synuclein-EGFP protein shows almost no difference in intensity as seen in the top three graphs. However in concert with GroEL it shows a clear increase. Notably as seen in the three bottom graphs, the kinetics of a-synuclein-EGFP is slowed a lot by the combination with our biobrick and pGroE7. This is not seen in the bacteria with only the substrate and pGroE7.
Figure 5. Results for a-synuclein-EGFP. Top graphs show fluorescene intensity divided by the start OD600 over 16 hours at 37 degrees celsius. The bottom graphs show the normalized values from the top graphs.
Results for the Tau0N4R-EGFP substrate protein
The last protein we tested was the Tau0N4R-EGFP construct. The top graph without our biobrick show a clear increase in intensity, and a small decrease in the folding rate as seen in the bottom graph. Interesting to note is that the bacteria with our biobrick and the pGroE7 slowed down the folding rate by a large margin. It is also clear that there is no decrease in the intensity at any point in the graph, and that this combination would maybe had increased up and over the substrate reference.
Figure 6. Results for Tau0N4R-EGFP. Top graphs show fluorescene intensity divided by the start OD600 over 16 hours at 37 degrees celsius. The bottom graphs show the normalized values from the top graphs.
Overview of our experimental design to archive the results shown below
A simple explaination on how we archived our experimental results is shown in figure 2.
Figure 2. The plasmids shown is the same as before, where A can be either of mNG-Aß1-42, EGFP-Aß1-42, a-synuclein-EGFP and Tau0N4R-EGFP. B is always pGroE7 and C is pSB4A5-GroES. The concentrations used for inducing the chaperone plasmids was 0.25 mg/ml L-arabinose, 200 ng/ml tetracycline. For the substrate plasmids we used 0.5 mM IPTG, and the substrate was induced 30 mins after the chaperone plasmids.
Results from the experimental measurements is shown below. The y-axis represent the fluorescence intensity at 520 nm. The excitation was done at 485 nm. The x-axis show the time for the measurements. The second set of graphs shown for each substrate represent the normalized values for the fluorescence intensity. This was analyzed because it represent the kinetic growth of the substrate proteins. As seen in the those graphs, the 50 % of max has been marked out as a dotted line. This makes it easy to interpret if the folding of the different substrate proteins was affected by the chaperone systems.
Results for the mNG-Aß1-42 substrate protein
As seen in the top three graphs our biobrick increases the intensity of the mNG-Aß1-42 protein. However a interesting notice is that in concert with the pGroE7 plasmid, it seem to slow the folding rate significantly as seen in the bottom graphs. A high concentration of GroES, with or without GroEL seems to be the best fit for this substrate protein. While a double-expressed GroES gene in concert with GroEL seems to hinder the production of the substrate more than helping it. This might be because mNG-Aß1-42 is fairly good at folding itself, and a very high level of co-expression, while also dealing with three antibiotics.
Figure 3. Results for mNG-Aß1-42. Top graphs show fluorescene intensity divided by the start OD600 over 16 hours at 37 degrees celsius. The bottom graphs show the normalized values from the top graphs.
Results for the EGFP-Aß1-42 substrate protein
The results for the EGFP-Aß1-42 protein looks quite different compared to the mNG-Aß1-42 protein. In this case our biobrick and the other two combinations show an increase in fluorescence intensity compared to the substrate alone. A interesting notice from the bottom three graphs is that our biobrick without the presence of GroEL slows down the folding rate significantly, and a decrease in intensity is not shown after 16 hours. Perhaps the EGFP-Aß1-42 protein folds better and stays correctly folded better with a low concentration of GroEL.
Figure 4. Results for EGFP-Aß1-42. Top graphs show fluorescene intensity divided by the start OD600 over 16 hours at 37 degrees celsius. The bottom graphs show the normalized values from the top graphs.
Results for the a-synuclein-EGFP substrate protein
The results for the a-synuclein-EGFP protein shows almost no difference in intensity as seen in the top three graphs. However in concert with GroEL it shows a clear increase. Notably as seen in the three bottom graphs, the kinetics of a-synuclein-EGFP is slowed a lot by the combination with our biobrick and pGroE7. This is not seen in the bacteria with only the substrate and pGroE7.
Figure 5. Results for a-synuclein-EGFP. Top graphs show fluorescene intensity divided by the start OD600 over 16 hours at 37 degrees celsius. The bottom graphs show the normalized values from the top graphs.
Results for the Tau0N4R-EGFP substrate protein
The last protein we tested was the Tau0N4R-EGFP construct. The top graph without our biobrick show a clear increase in intensity, and a small decrease in the folding rate as seen in the bottom graph. Interesting to note is that the bacteria with our biobrick and the pGroE7 slowed down the folding rate by a large margin. It is also clear that there is no decrease in the intensity at any point in the graph, and that this combination would maybe had increased up and over the substrate reference.
Figure 6. Results for Tau0N4R-EGFP. Top graphs show fluorescene intensity divided by the start OD600 over 16 hours at 37 degrees celsius. The bottom graphs show the normalized values from the top graphs.
Results for the mNG-Aß1-42 substrate protein
As seen in the top three graphs our biobrick increases the intensity of the mNG-Aß1-42 protein. However a interesting notice is that in concert with the pGroE7 plasmid, it seem to slow the folding rate significantly as seen in the bottom graphs. A high concentration of GroES, with or without GroEL seems to be the best fit for this substrate protein. While a double-expressed GroES gene in concert with GroEL seems to hinder the production of the substrate more than helping it. This might be because mNG-Aß1-42 is fairly good at folding itself, and a very high level of co-expression, while also dealing with three antibiotics.