Team:Nanjing-China/Experiments
Experiments
Preparation of polyP
Purpose:
To prepare a sufficient amount of pure polyP with suitable chain length and suitable for binding.
Experimental results:
1. Through bacterial synthesis, we obtained 839.28mg of polyP in the bacterial body.
2. After extraction, we obtained 489.89mg of pure polyP product.
3. Through the ethanol separation method, we finally obtained 214.67mg of polyP 45 with a suitable chain length that can be used in subsequent binding experiments.
4. We have obtained a set of methods that can efficiently produce polyP, and determined the yields of extraction and purification steps. The polyP plasmid is highly expressed in Citrobacter fujii. After being expanded in LB medium for 12 hours, it is inoculated into the wastewater medium. The bacteria can use the inorganic phosphorus in the synthetic wastewater to synthesize polyphosphate. The maximum yield is about 20mg/L.
5. We have clarified the interference factors and their effects in each link in the production of polyP, and thus determined the methods that can increase the yield of polyP. Our plasmid is resistant to kanamycin, so it grows predominantly in LB medium supplemented with kanamycin. At the same time, it is the only predominant species in wastewater medium due to the scarcity of nutrients, but in wastewater medium The number of reproduction is not much and more energy is used to synthesize polyphosphate, and the yield is the highest 14 hours after inoculation, and the prolonged time may lead to a decrease in yield and the decomposition of polyphosphate. Among them, 100ml of synthetic wastewater in a 200ml Erlenmeyer flask has the highest system output and the highest conversion efficiency. Almost all inorganic phosphates can be converted into polyphosphates under the condition of no other bacteria pollution and normal bacterial viability.
6. The content of polyP synthesized by CPP recombinant engineering bacteria at different culture time.
Figure 1 The change of polyP synthesis in engineering bacteria over time
As shown in Figure 1, at the beginning of the culture, the relationship between the amount of polyP synthesis in CPP and the time is close to linear growth, and the polyP production reaches its maximum after 16 hours.
+36GFP preparation
Purpose:
To prepare high-purity, super charged GFP(+36GFP) that can be used in binding experiments.
Experimental results:
We ligated the +36GFP target gene sequence to the PET29a vector to obtain the recombinant plasmid PET29a-+36GFP. The recombinant plasmid was transferred to the competent cell BL21 (DE3), and the final concentration of 1mM IPTG was induced at 16°C overnight for subsequent use of protein purification. In the experiment of purifying +36GFP, we first use Ni column to purify the target protein with histidine tag, and then use ion exchange column to purify +36GFP (to achieve the purification of positively charged protein). The SDS-PAGE detection results of the protein purified by the above two columns are shown in the figure below:
Figure 2 SDS-PAGE diagram of each solution during purification using ion exchange column.
Figure 3 SDS-PAGE detection results of +36GFP and spidroin after purification.
In the lane corresponding to the sample solution, a band near 30kDa appeared, and no band of 30kDa appeared in the effluent of the sample, indicating that +36GFP in the sample solution can bind to the ion exchange column. In the corresponding lane of Binding Buffer, there is no band of 30kDa, which means that +36GFP cannot be eluted by Binding Buffer, and the sample contains less contaminated protein. In the effluents of eluate 1 and eluate 2, obvious bands appeared near 30kDa, indicating that +36GFP was eluted and +36GFP was successfully purified. Combined with the electrophoresis results of +36GFP after purification in Figure 3, there is a thicker band near 30kDa, which further shows that +36GFP was successfully purified.
+36GFP-SP fusion protein preparation
Purpose:
To prepare large amounts of high-purity, super charged GFP(+36GFP) fused with SP that can be used in binding experiments.
Experimental results:
We attached +36GFP-SP fusion protein gene sequence to PET28a vector and obtained the recombinant plasmid PET28a-+36GFP-SP, which was transformed to the competent cell BL21 (DE3) and induced by 1M IPTG at 25℃ overnight for later protein purification. The protein purification result shows that +36GFP-SP fusion protein produced by recombinant plasmid PET28a-+36GFP-SP can interact with Ni column and led to the successful purification. The SDS-PAGE detection results of the protein purified by the two kinds of columns are shown in the figure below:
Figure 3.1 Result of +36GFP-SP SDS-PAGE
In reference to figure 3.1, bands of 70kDa appeared in both total protein track and supernatant track, while no band of 70kDa appeared in the precipitation track. This indicates that +36GFP-SP fusion protein gene was successfully expressed by BL21 and most +36GFP-SP fusion protein existed in supernatant after cell disruption, which made it of value for the following purification step. Moreover, combined with the electrophoresis results of +36GFP-SP after purification in Figure 1, a weak band near 70kDa appeared, which further indicated the success of +36GFP-SP purification.
The combination experiment of +36GFP and polyP
Purpose:
To test whether the combination of polyP and +36GFP will increase the thermal stability, and to explore conditions for the subsequent binding of polyP and spidroin.
Experimental results:
We first determined the phase separation boundary where +36GFP binds to polyP, and selected the corresponding polyP and +36GFP concentrations in the phase separation boundary to perform binding experiments.The results of our experiments to find the boundary are shown in the figure3.1 below. The red dots show the binding condition of the two within the boundary. As can be seen from the fluorescence diagram on the left, the two can be combined into a tight substance at a specific concentration within the boundary.
Figure 3.1 In vitro phase separation of +36GFP driven by polyP
PolyP (Sigma, Type45, Item S4379) with a final concentration of 3200μM and +36GFP (with an electrostatic charge ratio of 32) with a final concentration of 2.78μM are mixed in equal volumes, and the two are combined in the solution through electrostatic interaction to obtain a polyP-GFP solution. Next, we characterize the stability of the fluorescence of +36GFP before and after the polyP is combined with the degree of decay of the GFP fluorescence intensity with time before and after the combination, and the effect of different temperatures on the fluorescence intensity.
1.GFP fluorescence intensity changes with time before and after binding at room temperature.
Figure 4 Fluorescence of polyP-GFP, GFP and control solution after 3h, 7h, 24h, 48h combination
As shown in Figure 4, the fluorescence of GFP will decrease significantly with time. Under the same conditions, +36GFP combined with polyP has higher fluorescence brightness than +36GFP without polyP, especially after 48h, the fluorescence of +36GFP combined with polyP is stable and the fluorescence brightness is high, which shows that polyP can stably protect GFP, prolong the fluorescence time of GFP. Therefore, we can preliminarily speculate that polyP can protect the stability of GFP fluorescence.
Next, 5.56μM +36GFP and 10μM +36GFP were mixed with different concentrations of polyP in equal volumes, respectively, and the fluorescence intensity was detected after 0h, 3h, 24h, and 48h incubation.
Figure 5 The effect of different polyP binding concentration on GFP fluorescence intensity (0~48h)
As shown in Figure 5, under the same binding time, as the binding concentration of polyP increases, the fluorescence stability of polyP increases, the fluorescence intensity increases significantly, and then slowly increases. When the binding concentration of polyP is low, GFP is excessive and polyP binds to part of GFP. At this time, increasing the binding concentration of polyP will increase the GFP binding to polyP and increase the fluorescence intensity.
All the above are the results we obtained from our in vitro binding experiments. We also tried binding GFP with polyP in vivo, and the results are shown in the figure5.1. The results showed that +36GFP and polyP could be synthesized simultaneously in vivo and directly combined in bacteria.These results can help us better apply the experimental results to the production of our products.
Figure 5.1 +36GFP binding to polyP in vivo
2. Thermal stability of GFP fluorescence intensity before and after polyP binding.
(1) PolyP (6400μM) and +36GFP (5.56μM) are mixed in equal volume, in the phase boundary of liquid-liquid separation, incubated at room temperature for 30min, and then 25, 30, 40, 50, 60, 70, 80, 90, 100 Incubate at ℃ for 10 min. After cooling, the fluorescence intensity is detected by the microplate reader.
Figure 6 The influence of temperature on the fluorescence intensity of GFP
After GFP is combined with polyP, the fluorescence intensity after incubation at different temperatures is greater than that of GFP that is not combined with polyP, indicating that the combination of polyP and GFP can protect GFP, and the relative thermal stability of GFP is increased. To further determine the influence of polyP on the thermal stability of +36GFP, we did the following experiment.
(2) The final concentration of polyP (0,100,400,800,1600,3200μM) and the final concentration of +36GFP (2.78μM) were mixed in equal volume, incubated at room temperature for 30min, and then 25,30,40,50,60,70,80,90,100℃ After incubating for 10 minutes, after cooling, the fluorescence intensity is detected by the microplate reader
Figure 7 The effect of different final concentrations of polyP on the fluorescence stability of +36GFP
As shown in Figure 7, with the increase of polyP binding concentration, the fluorescence intensity of +36GFP increases, and when the fluorescence intensity decays to 50%, the corresponding temperature Tm also rises significantly (Figure 8), indicating that the combination of polyP and +36GFP can increase+ The thermal stability of 36GFP.
Figure 8 The effect of different concentrations of polyP on the Tm value of +36GFP
3. The change of fluorescence intensity in protein denaturation solution before and after +36GFP combined with polyP.
+36GFP+polyP: polyP (0,200,800,1600,3200,6400) and +36GFP (5.56μm) mixed in equal volume (50ul+50ul), respectively incubated at room temperature for 30min, then mixed with denaturing solution (100ul) and incubated for 30min, enzyme label The instrument detects the fluorescence intensity. (1X denaturing solution; 4M urea, 0.5% SDS, 3M guanidine hydrochloride. 2X: 8M urea, 1% SDS, 6M guanidine hydrochloride)
Figure 9 The effect of different binding concentrations of polyP on the fluorescence intensity of +36GFP in denaturing solution