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. The red segments of the plasmids represents the antibiotivs. The green segments represent the promotors and the yellow represent the origin of replication.
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 mNG-Aß1-42, EGFP-Aß1-42, α-synuclein-EGFP and Tau0N4R-EGFP.

Overview of our experimental design to archive the results shown below

A simple explanation on how we achieved our experimental results is shown in figure 2.
Bacteria containing plasmids, see protocol, were induced with IPTG, L-arabinose and Tetracycline in LB-growing media until desired OD was achieved. Then the bacteria samples were placed in 96-well plate that ran for 16 hours in 37 degrees celsius in a spectrophotometer. Measurements were conducted every fifteen minutes. As seen in figure 2, four combinations were introduced into E.coli (BL21). Substrate (A) , substrate and our biobrick (A +B), substrate, our biobrick and GroE (A+B+C), substrate and GroE (A+C). A can be either mNG-Aß1-42, EGFP-Aß1-42, α-synuclein-EGFP and Tau0N4R-EGFP. B is always pGroE7 and C is pSB4A5-GroES.

Figure 2. The plasmids shown is the same as before. 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

Results from the experimental measurements are shown in figure 3. The y-axis represents the fluorescence intensity at 520 nm. The excitation was done at 485 nm. The x-axis shows 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 represents the kinetic growth of the substrate proteins.
As seen in 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, an interesting notice is that in concert with the pGroE7 plasmid, the results create the impression that the folding rate slows significantly, as shown in the bottom graphs.
A high concentration of GroES, with or without GroEL seems to be the best fit for this substrate protein. A double-expressed GroES-gene together with GroEL seems to prevent the production of the substrate more than helping it.

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. An interesting notice from the bottom three graphs is that our biobrick without the presence of GroEL slows down the folding rate, and a decrease in intensity is not shown after 16 hours.

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 α-synuclein-EGFP protein shows almost no difference in intensity which can be seen in the top left graph. However, in concert with GroEL it shows a clear increase. Same thing goes with co-expressing GroES with GroE, though it increases more slowly compared to only the substrate.
Notably as shown in the three bottom graphs, the kinetics of α-synuclein-EGFP is slowed down a lot by the combination with our biobrick(GroES) and pGroE7(GroE). 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 tested was the Tau0N4R-EGFP construct. The top graphs without our biobrick (the blue lines) 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(GroES) and the pGroE7 (GroE) 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 this graph. The same results can be applied for the plasmid with only GroE.

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.

Discussion and conclusion

mNG-Aß1-42: As mentioned, the folding rate slows down when pGroE7 is present. The combination with a GroES and GroE seems to prevent the production of the substrate. An explanation for this could be that mNG-Aß1-42 is fairly good at folding itself and that the presence of chaperones may instead inhibit the folding.
The best fit for the substrate protein is our biobrick, or a high concentration of GroE, which means that our biobrick and GroE-plasmid is in this case best for increasing the amount of substrate and give the highest folding rate.

EGFP-Aß1-42: As seen in figure 4, when adding chaperones to the systems, the concentration of substrate increases compared to without adding GroES or GroE. The graph presenting the results from our biobrick has not reached its maximized concentration of the substrate. Potential future studies is needed in order to find out the maximized concentration of the substrate. This could be done with an increased experimental time.
The graphs presenting the folding rate of each expressed system shows little difference.

α-synuclein-EGFP: When expressing α-synuclein-EGFP with GroES there is little difference in concentration compared to expressing α-synuclein-EGFP without a plasmid-chaperone. With GroE the concentration of the substrate greatly increases compared to all other expression systems. The kinetics of the folding rate for α-synuclein-EGFP expressed with GroE and GroES is noticeably decreased. It seems that when GroEL is expressed with an increased concentration of GroES it decreases the folding rate of this substrate. If GroEL is not expressed though, there is no difference in the folding rate.

Tau0N4R-EGFP: There is no decrease in intensity for Tau0N4R-EGFP combined with chaperones, and a study with longer experiment time can be useful to study the maximum concentration. The folding rate for all of the substates with chaperones slows down, but with difference in how much. In presence with GroES the folding rate is higher than with GroES and GroE co-expressed. With GroE, the folding rate is in between the others.