Team:Nanjing-China/Engineering

At the suggestion of manager from Leqi textile, we mimicked strong electrostatic interactions in the binding between spidroin and polyP. Since the modeling parts indicated that amino acids like arginine, lysine and histidine contribute mostly to the electrostatic interactions, we tried similarly to mutate the primary sequence of spidroin to change its charges.

Build&Test: Spidroin Granules

Plasmid information

We changed the negatively charged amino acids in the non-helical and folded loop structures of spider silk proteins to lysine (the most positive amino acid) without affecting the main structure of the protein. It is positively charged to combine with the negatively charged polyphosphate through electrostatic force. In future experiments, the properties of spider silk proteins can also be improved through the combination of positively charged spider silk proteins and negatively charged inorganic molecules. Or through the fusion of spider silk protein and positively charged green fluorescent protein, the whole protein is positively charged, and at the same time, it combines the advantages of spider silk protein and green fluorescent protein, so that the position of this protein has green fluorescence. It can be clearly seen during separation or cell positioning, which is convenient for the observation of experimental results. It also has the good properties of spider silk protein. After combining with other organic and inorganic substances, it can be made into spider silk, which has good toughness and strength.

The negative charged amino acid in the non-helix and folded ring structure of spidroin was modified to lysine (the most positive electric amino acid) to make the spidroin positively charged without affecting the main structure of the protein. SP1 mainly modifies N-terminal and C-terminal.

Plasmid function verification

In subsequent experiments, we combined the spider silk protein green fluorescent protein with polyphosphate, which has better thermal stability, PH stability and better mechanical resistance. The spider with positive electricity Silk protein can be easily and tightly combined with negatively charged molecules, which is convenient for studying the physical and chemical properties of different organic-inorganic hybrid materials. In the experiment of purifying spider silk protein, we first use Ni column to purify the target protein with histidine tag, and then use ion exchange column to purify spider silk protein (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:

Figure 1 spidroin SDS-PAGE test result

Remarks: BL21 refers to the transfer into the empty PET29a vector without the target gene, and the specific whole protein is obtained by inducing expression.

Figure 2 purified +36GFP、spidroin SDS-PAGE test result

As shown in Figure 1, in the whole protein, Sp and Sp1 have bands around 40KDa, indicating that Sp, Sp1 and Sp2 can all be expressed in E. coli. Among them, Sp2 has a more obvious band around 40KDa, indicating The expression level of Sp2 in E. coli is relatively large. In the precipitate, Sp2 appeared as a band around 40KDa, but there was no corresponding band in the supernatant, indicating that Sp2 was mainly present in the precipitate. The bands of Sp and Sp1 are not obvious in the whole protein and supernatant, indicating that the expression level is low.

In order to further verify the expression of Sp, Sp1 and Sp2, we performed SDS-PAGE again. As shown in Figure 2(A), the whole protein of Sp2 appears in a corresponding band near 40KDa, indicating that Sp2 can be expressed in the bacteria. In the precipitation, Sp2 showed a band near 40KDa, but there was no band near 40KDa in the supernatant, indicating that Sp2 protein mainly exists in the precipitate. As shown in Figure 2 (A), the supernatants of Sp and Sp1 have bands around 40KDa, indicating that Sp and Sp1 can also be expressed in the bacteria, but the expression level is low and the column hanging effect is poor.

Learn: Learnt from interviews with Prof. Tian&Prof. Cao

We interviewed Mr. Cao and Mr. Tian from College of engineering and applied sciences. Prof. Tian gave us suggestions on protein purification and stressed the importance of buffer for successful protein elution. Besides, professor Cao pointed out that reconstructing spidroin may affect its folding. Therefore, he proposed us a new idea and inspired us to combine spidroin with + 36GFP to form a new fusion protein, and then hybrid the fusion protein with polyP.

Redesign&Rebuilt: We built a new part and proved it worked as expected

Inspired by these professors, we tried to fuse GFP and spidroin.

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 Result of +36GFP-SP SDS-PAGE

In reference to figure 3, 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.

More experiments are still continuing.

More information about our experiments

Related plasmids synthesized by polyP

Plasmid information

Natural function of part: The polyphosphate kinase in Citrobacter freundii ATCC 8090 is responsible for its intracellular inorganic polyphosphate (polyP) production via reversibly catalyzing the transfer of terminal phosphate from ATP to a growing polyP chain.

Wild-type Citrobacter freundii ATCC 8090 was purchased from China Center of Industrial Culture Collection (CICC, China). The ppk1 gene was acquired by PCR. This organism is our chasiss, in which its native PPK1 will be overexpressed with a plasmid of medium-copy numbers.

Although PPKs from E. coli and C. freundii shares 96% amino acids’ identity, the C. freundii PPK has a glutamate and a lysine residue in positions 327 and 328, where in E. coli they are substituted by much less strongly charged alanine and glutamine residues, respectively. Although these natural occurred mutation sites found in C. freundii PPK are distant from the enzymes’ active site, they lie in the interfaces among monomers of the PPK tetramer. Benefit from this difference, a dramatic increase of intracellular polyP accumulation can be achieved with C. freundii.

Plasmid function verification

The polyP plasmid is highly expressed in Citrobacter fujii. It is inoculated into wastewater culture medium after 12 hours of expanded culture in LB medium. The bacteria can use the inorganic phosphorus in synthetic wastewater to synthesize polyphosphate, the largest The output is about 20mg/L. 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. Finally, we obtained 839.28mg of polyP in the bacteria through bacterial synthesis; after extraction, we obtained 489.89mg of pure polyP; through the ethanol separation method, we finally obtained 214.67mg of polyP 45 with suitable chain length that can be used in subsequent binding experiments.

+36GFP plasmid

Plasmid information

The nucleotide sequence of GFP (+ 36) was biosynthesis of Qingke, using PUC57 with ampicillin resistance. The N-terminal restriction site was KPN I, the C-terminal site was hind III, and the host cell was E.coli Top 10. GFP green fluorescent protein (GFP) is improved by adding 36 positive charges, making it easier to combine with negatively charged proteins through electrostatic force. Because GFP has intrinsic green fluorescence, it is easier to be located in cells and tissues, not only can determine the distribution of negatively charged proteins, but also, to a certain extent, can indicate the electrical properties and structural changes of binding proteins. Positively charged green fluorescent protein (GFP) provides negatively charged molecules with a way to change their electrical properties, bind to negatively charged molecules and label negative proteins.

Plasmid function verification

After comparison and verification, the synthesized nucleotide sequence is translated into protein, which is consistent with the expected sequence. We ligated the +36GFP target gene sequence to the PET29a vector to obtain the recombinant plasmid PET29a-+36GFP. The recombinant plasmid was transformed into the competent cell BL21 (DE3), and the final concentration of 1mM IPTG was induced at 16°C overnight for subsequent 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: