Team:WHU-China/Results
Cell-Free Quorum Sensing
1. To reconstitute the basic logic of las and rhl systems in E. coli lysates
2. To screen quorum sensing inhibitors from a library of natural products
Quenching Module
AHLase Expression
1. To express lactonase AiiA and acylase AiiC, PvdQ and AiiO in E. coli BL21(DE3)
2. To test their degradation efficiency
Sensing Module
TEV protease expression
1. To express TEV protease with and without His-tag
Primary Chemokine selection
1. To test the efficiency of inducing cell migration of each chemokine
2. To calculate chemotaxis index of each chemokine and choose appropriate chemokines
Toxin-Antitoxin Systems
1. To clone toxin and anti-toxin pairs from E. coli K-12 MG1655
2. To test their efficiency of preventing HGT and choose a best TA system
Cell-Free Quorum Sensing
1. Four plasmids to reconstruct quorum sensing systems were constructed
2. A plasmid for negative control in the drug screening process was constructed
Quenching Module
AHLase Expression
1. AiiC and AiiA genes were constructed, transferred and expressed in E. coli BL21(DE3), but further
data was required; PvdQ and AiiO were unavailable
Sensing Module
TEV protease expression
1. TEV protease with and without His-tag was successfully constructed (confirmed by colony PCR), but
there was no expression attempt
Primary Chemokine selection
1. Successfully obtained the migrated number of THP1 cells induced by each chemokine (shown in
Figure 6 to Figure 12)
2. Determine the optimal chemokine for THP1 (at a concentration of 10nm/L)
Toxin-Antitoxin Systems
1. Successfully constructed MazE and MazF (confirmed by colony PCR), while the attempts to clone
PemI, PemK, HipB, and HipA failed
All the plasmids in Cell-Free Quorum Sensing need two devices to be inserted, and the expected efficiency is low. After nearly two weeks of unsuccessful digestion and ligation, Hieff Clone® Plus Multi One Step Cloning Kit was suggested by our advisor as an alternative. With Sangon Biotech sending primers for us, we used PrimeSTAR® GXL DNA Polymerase to add homologous arms flanking the genes synthesized by IDT and GenScript (Figure 1). Using Hieff Clone® Plus Multi One Step Cloning Kit, the ligation (50℃, 20 minutes) was performed, as well as later transformation.
Figure 1. PCR amplification by PrimeSTAR® GXL DNA Polymerase
Successfully, several right bands were observed by colony PCR (Figure 2), and corresponding single colonies were inoculated to 10 mL LB media and incubated overnight (37℃, 220 rpm).
Figure 2. Colony PCR for correct single colonies
Plasmids were extracted from overnight cultures, and quantified by Thermo Scientific™ NanoDrop™ One. Later the five plasmids were digested by EcoRI and SpeI, and right but dim bands were observed. For final validation, all five plasmids and pSB1A3 sequencing primers (BBa_G00100 and BBa_G00101) were sent to Sangon Biotech for gene sequencing.
In summary, five plasmids with confirmed inserted sequences were constructed, and further experiments based on these five plasmids will be implemented after this season.
The main goal of our quenching module is inserting the genes encoding quenching enzymes into expression vectors and transferring them into host bacteria to express the enzymes. With the help of GenScript, we got the AiiA gene synthesized and inserted into pET-28a (+) directly. And to verify the correct ligation of the gene, we used EcoR1 and Pst1 to digest the plasmids and got the expected result, which was also the same as the company’s (Figure 1). We noticed that the original plasmid band (over 12kb) is much larger than expected(6-7kb), but it may be due to the complex conformation of the pET-28a(+)(which we asked our instructor for explanation). And as there are two bands in the lane of digestion products (one of which is dim) of correct size, we can basically conclude that the expression vector is successfully constructed. And we have successfully transformed it into E. coli BL21(DE3) (not shown). But this vector still needs to be further confirmed by colony PCR and gene sequencing. And next season, it can be used directly to get the enzymes we want.
Figure 3. Enzyme digestion for correct plasmids (pET-28a(+)-AiiA. L1: plasmids got from company. L2: digestion products of plasmids synthesized by company.
For the gene fragment encoding AiiC we got from IDT, we digested it and the pSB1C3 vector from iGEM kit with EcoR1 and Pst1(37°C, overnight). And then we used Rapid DNA Ligation Kit, Beyotime (D7002) to ligate the gene with the pSB1C3 vector. After the transformation of it into E. coli BL21(DE3), we extracted the plasmids and digested them again with the same enzymes to check if the result was positive. And the electrophoresis showed the target band of the recombinant plasmid but only one trailing band in the lane of the digestion products (Figure 2). So, we proposed that it may be due to the unsuccessful separation of the AiiC gene and the vector fragment after digestion. Thus, it also needs colony PCR and gene sequencing for further verification.
Figure 2: Enzyme digestion for correct single colonies carrying pSB1C3+AiiC. L1: plasmids extracted from BL21(DE3) colonies. L2: enzyme digestion products of the plasmids extracted.
However, synthesis of pvdQ was failed due to its high GC content and failure occurred on AiiO as well for unknown reason. And we failed to obtain PvdQ from Pseudomonas aeruginosa PAO1 directly by colony PCR. So, unfortunately, we can’t conduct experiments with PvdQ and AiiO this year. And we may continue to experiment with them in next season but may change them for other genes that are easier to get.
The TEV protease with His-tag is planned to be used in the subsequent detection of the effectiveness of the secretion restriction device, and the TEV protease without His-tag is used to conveniently remove from the system after cleaving the N-terminal of the chemokine. The TEV protease gene was digested with EcoR1 and Pst1 and then ligated to pSB1A3 with the same operation. The colony PCR result of the positive colonies after the ligation product is transformed shows that the connection is successful. Due to the failure of the related parts transformation, neither of the two TEV proteases tried to be expressed in the bacteria.
Figure 4. Colony PCR confirmed the success of the connection
In our design, we want to use chemokines to recruit inflammatory cells, mainly neutrophils and monocytes to clear the pathogens. We had chosen 7 chemokines, CXCL1, CXCL2, CXCL3, CXCL8, CCL2, CCL3 and CCL8, which may be suitable for our project. We planned to select 2 to 4 chemokines with high chemotaxis ability at appropriate concentrations from these chemokines to express in our engineered bacteria. We used differentiated HL-60 cell line as the model of neutrophils and THP1 cell line as the model of monocytes. To evaluate the chemotaxis of the 7 chemokines to THP1 cells, transwell migration assay was conducted, each condition with 3 parallel experiments and each transwell board with at least 3 parallel controls. The migrated THP1 cells in the lower chamber were counted by flow-cytometry. The primary results are shown in the form of scatter diagram [Figure 5].
Figure 5. An example of primary cell counting result from flow-cytometry, in which CCL2 was examined. The red marked dots in the red frame are records of THP1 cells in the lower chamber. The number of these dots indicates the number of THP1 cells in the lower chamber.
The number of cells in each lower chamber was then collected according to these scatter diagrams and analyzed. Analyzed results are shown in Figure 6 to Figure 12. The results suggested that at the concentration of 10 nmol/l, 3 chemokines from CCL family have the potential to attract THP1 cells, especially CCL2, which shows statistical significance(p < 0.05). However, 4 chemokines from CXCL family do not show a potential to attract THP1 cells and they even have a tendency to inhibit the cells’ migration at the concentration of 10 nmol/l. Moreover, the data of several chemokines like CCL3, CXCL8 and CXCL3 shows great standard error, which indicates their unstable performance of chemotaxis at the concentration of 10 nmol/l.
Figure 6. Chemotaxis of CCL2 to THP1(shown by cell number). Chemokine concentration is 10 nmol/l.(n=3)
Figure 7. Chemotaxis of CCL3 to THP1(shown by cell number). Chemokine concentration is 10 nmol/l.(n=3)
Figure 8. Chemotaxis of CCL8 to THP1(shown by cell number). Chemokine concentration is 10 nmol/l.(n=3)
Figure 9. Chemotaxis of CXCL1 to THP1(shown by cell number). Chemokine concentration is 10 nmol/l.(n=3)
Figure 10. Chemotaxis of CXCL2 to THP1(shown by cell number). Chemokine concentration is 10 nmol/l.(n=3)
Figure 11. Chemotaxis of CXCL3 to THP1(shown by cell number). Chemokine concentration is 10 nmol/l.(n=3)
Figure 12: Chemotaxis of CXCL7 to THP1(shown by cell number). Chemokine concentration is 10 nmol/l.(n=3)
The chemotactic index (CI) of each chemokine was calculated and displayed in Figure 13. It is obvious that CI of CCL2 was the highest, and CCL3 comes the second and CCL8 the third.
Figure 13. Chemotaxis of 7 chemokines to THP1, shown by chemotactic index. Chemokine concentration is 10 nmol/l.(n=3)
According to the results all above, CCL2 was the best chemokine to attract THP1 cells, because CCL2 has the highest CI and its enhancement of cell migration has statistic significance. Although the CI of CCL3 is just a little lower than that of CCL2, its performance of chemotaxis is unstable and shows no statistic significance compared with the control. Therefore, the result suggests CCL2 to be the one we should select to use.
Because of time limitation and failure on Hl-60 cell culture, the transwell migration assay on differentiated HL-60 cells was not conducted. Moreover, the concentrations of all chemokines are equal to 10 nmol/l. The results when the concentration differs remains unknown. Next year, further experiments may include these tests.
Toxin-antitoxin systems are used to prevent horizontal gene transfer in our design. We planned to put two pairs of toxin and antitoxin gene separately on two different plasmids. To achieve that, on each plasmid the toxin comes from a system while the antitoxin comes from another. In order to get the toxin and antitoxin genes separately, we decide to use colony PCR. Here shows our colony PCR results of MazE and MazF gene from Escherichia coli K-12 MG1655 [Figure 14 and Figure 15]. The bright band in front of 2oobp band was identified as primer dimers for defective design of primers.
Figure 14. Agarose gel electrophoresis results of MazE colony PCR. The size of MazE CDS is 264bp.
Figure 15. Agarose gel electrophoresis result of MazF colony PCR. The size of MazF CDS is about 333bp.
We also extracted the pET-28a, preparing to use it as the carrier of our toxin and antitoxin genes[Figure 16]. The bands of pET-28a extracted is in front of 4000bp, smaller than the size of itself(5.4kb). This may be caused by the supercoiled conformation of the extracted pET-28a.
Figure 16. Agarose gel electrophoresis of extracted pET-28a. The size of pET-28a is about 5.4kb.
The colony PCR of PemI, PemK, HipB and HipA all failed. This may be caused by unsuitable annealing temperature or poor design of primers. Solving this problem requires more experiments. However, because of time and conditional limitation, we neither made more trials nor did any further molecular cloning experiments. These further works are planned to be conducted next year.
