Results
Result: Specific gene circuit:
Module I:The growth-promoting effects of glucose dehydrogenase(GDH)
Several studies have proven that overexpression of GDH does have growth-promoting effects on bacteria. In our experiments, E. coli was used as the chassis species and the gene sequence of GDH came from the genome of E. coli K12 strain.
Former studies only applied SDS-PAGE Gel electrophoresis in order to confirm the existence of GDH, which merely provides the information of molecular weight. In order to validate the existence of GDH precisely, two tags (6xHis and V5) were added at the C-terminal of the protein, which enabled further experiments including purification and western blot. Eventually, we proved the expression of our recombinant plasmid (Fig. 14a) in E. coli through western blotting (Fig. 1).
a) Design of the experimental plasmid.
b) A western-blot image of the overexpressed GDH, from left to right are: bacteria with the recombinant plasmid, bacteria with the wild-type plasmid and wild-type bacteria (no plasmid transformed).
c) the growth curve with a dilution ratio of 1:10, y-axis represents OD600 and x-axis represents time. Through all the experiments, M9 media is used. The original OD600 is 1.60.
d) The bacteria growth curve after a dilution of 1:50, original OD600 is 0.58.)
2. Experiments in labs are always carried out with antibiotics present. Therefore, to imitate the antibiotic-free condition in industrial procedures, we improved our protocol. Firstly, we used M9 media with equal IPTG added to ensure a reliable outcome. No antibiotics was added to the media. Cultures were incubated overnight in LB media (liquid) with Ampicillin, and OD600 of all cultures were adjusted to the same value using an UV Spectrophotometer. We then diluted the cultures to 10 or 50 times using antibiotic-free media and incubated them in a shaker (37℃, 200rpm). By measuring the OD600 value of samples taken under proper timing, growth curves of different strains could thus be drawn (Fig. 9c and Fig 9d). Conclusively, we found that the strain with GDH gene grew faster than others, while the wild-type one took the second place. The strain with an empty vector transformed grew the lowest, which behaved as predicted, for an additional metabolic burden was added. These results showed GDH does promote the growth of bacteria.
Thus far, the verification of the ability of promoting bacteria’s growth of GDH had been finished. However, our final goal was to make the strain with GDH grows faster than those without a target gene, and becomes the dominant population of the system accordingly. Combining previous works and our data, some discussions are made as follows. We can see from Fig. 9c and 9d that overexpressed GDH helped in the increase of K value, while previous articles showed that dry weight of those with GDH overexpressed accumulated more during the same amount of time. Therefore, we could conclude that overexpressed GDH can improve the metabolic efficiency of bacteria, and such a character has the ability to resist the problem of target strain’s degeneration caused by the unequal distribution of plasmids. Meanwhile, in a fermentation system with vast amount of strains, those with higher metabolic efficiency can be seen as more “advantageous”. However, to what extent can the overexpression of GDH resist the trend of degeneration of strains with target genes remains unknown. The key to this problem is to validate the ability of GDH in keeping target genes. Hence, two tasks are needed to be carried out in this module: 1. Verify if GDH has the ability to keep target genes. 2. Build a persuasive model to answer the question stated above (To what extent can the overexpression of GDH resist the trend of degeneration of strains with target genes?).
Module II: pchBA producing salicylic acid
In this module, we used the part BBa_J45319 given by iGEM authority to transform E. coli strain BL21(DE3). Salicylic acid was confirmed generated after several times of induced expression with IPTG and liquid chromatography testing of the supernatant. The harvest was 10mg/L, more or less (Fig. 10a). For the convenience of further testing, we also drew a standard curve (Fig 10b) of salicylic acid’s concentration with a series of standard salicylic acid samples of which the concentration was known.
b) The standard concentration curve of salicylic acid
Module III: Salicylic acid and emrR binding promoter binds emrR protein competitively
This module is a transforming unit of the whole gene circuit, through which the signal of salicylic acid is transformed to the advantage in bacteria growth. The point of this module is to verify the inhibition effect of the repressor protein and the disinhibition effect of salicylic acid. Thus, we designed a part which expresses the repressor constantly, and an mcherry gene is placed downstream of the emrR binding promoter. Theoretically, this mcherry can only be expressed under the circumstance that salicylic acid is added to the media.
Figure 10 a. The expression of emrR prevents the initiation of PemrR, but the protein changes its configuration after binding with salicylic acid and leaves the promoter region, thus enables the expression of downstream proteins (in this case, mcherry). The promoter of emrR is a constant promoter, acquired from E. coli, originally responsible for the initiation of GADPH gene. When salicylic acid exists, red florescence can be observed. b. Control strain under white light, no salicylic acid added. c. Control strain under green light excitation, no salicylic acid added. d. Experiment strain under white light, SA conc. is 5mg/L. e. Experiment strain under green light excitation, SA conc. is 5mg/L.
The result of our experiment showed very low expression level of RFP when there was no salicylic acid in the media. When a concentration of 5~10mg/L salicylic acid was added to the media, the bacteria showed a peak level of red florescence intensity (induction time: 4h). The florescence intensity had not risen significantly when the concentration of salicylic acid was further increased. Meanwhile, as the concentration of SA increased, growth of the bacteria seemed to be repressed. We also found that cultures incubated in the 96-well plates did not show significant difference between control and experiment, but visible difference was observed when they were incubated in 10ml tubes. This probably due to the different growing environment of E. coli, indicating that the induction procedure of salicylic acid needs relatively large incubating space.
In conclusion, salicylic acid induction needs larger space, while emrR is highly sensitive when binding with SA, for that the peak level of expression is reached under very low SA concentration. However, the peak level is indeed not very large, which would possibly make the growth promoting effect not obvious, for a quite low level of SA would enable the expression of GDH keeps at a constant (peak) speed, which is quite low as well. Consequently, the harvest of SA would not obviously increase. Possible optimizations may focus on these following aspects: 1. Since the key problem is the low expression level of the growth promoting factor (GDH) and realizing that the RBS of emrR binding promoter from E. coli genome is not efficient, we could replace it with another RBS with higher ribosome binding efficiency. 2. Low expression level may ought to the fact that the circuit shown in Fig 10 is sensitive to salicylic acid. Thus, increase the level of expression of emrR could possibly decrease the sensitivity.