Team:GA State SW Jiaotong/Results
Plasmid Construction
Four new composite parts were designed or constructed in the project, as shown in Figures 1-4 below. They were either ordered directly from Qinke and Genscript or constructed by ourselves using enzyme digestion and Gibson Assembly. Besides this, as shown in Figure 5, we also applied codon optimization to a GUS gene to improve the transformation efficiency of this gene into the well-defined species Symbiodinium kawagutii.
Figure 1. DNA sequence map of pPICZαA-Gas6 plasmid.
This plasmid was synthesized by the company Qinke directly, which contained Gas6 protein encoding gene, AOX1 promoter, BleoR, and the high copy origin sequence.
Figure 2. DNA sequence map of pNW33N-EGF plasmid.
This plasmid was designed to be synthesized by adding an EGF sequence to the pNW33N+T7 plasmid by the company Qinke. In this process, restriction enzymes HindIII and SacI were used. This plasmid contained EGF coding sequence, CmR, and Pbr322 origin sequence.
Figure 3. DNA sequence map of pNW33N-Gas6 plasmid.
This plasmid was designed to be synthesized by restriction cloning between Pnw33n+T7+EGF and pPICZαA+Gas6 in GSU’s lab. In this process, restriction enzymes HindIII and SacI were used. This plasmid contained Gas6 coding sequence, CmR, and Pbr322 origin sequence.
Figure 4. DNA sequence map of the suicide switch.
This plasmid was designed to synthesize the Gibson assembly. In this synthesis process, the origin, Pet28a-TRP plasmid, was linearized twice. Genes Hok and Sok were assembled with linear plasmid after each linearization. At last, this plasmid contained Hok, Sok, Cmr, Pbr322 origin. Bacteria transformed by this plasmid can only survive in a medium with Lactose and Tryptophan.
Figure 5. Codon optimized GUS gene.
We used codon optimization to revise the DinoIII-GUS plasmid in hopes that it would make transformation into the Symbiodinium kawagutii more effective.
pNW33N+T7+Gas6 Plasmid Construction
We ordered the pNW33N+T7 backbone plasmid from the Genscript company. We then synthesized this pNW33N+T7+Gas6 plasmid by restriction cloning between pNW33N+T7+EGF and pPICZαA+Gas6. Restriction enzymes HindIII and SacI were used in this process. At the very beginning, the Gas6 coding sequence was amplified by PCR using Gas6+signal_top primer and Gas6+signal_bottom primer. Then, Gas6 coding sequence and pNW33N+T7 were cut by HindIII and SacI. After that, two sequences were linked through ligation. Finally, the ligated plasmid was transformed into Bacillus subtilis WB600 and single colonies were obtained after overnight culture on the LB + CMR plate. After comparing the bacterial growth status on the LB plate with antibiotics to the bacterial growth status on the LB plate without antibiotics, the selection works.
Fig. 6 Transformation results of E.coli BL21 transformed with pNW33N-Gas6.
The control group: pNW33N+Gas6+Lac promoter (LB). The experimental group: pNW33N+Gas6+Lac promoter (LB+CM). Many single colonies were obtained after overnight incubation according to the experimental group.
Single colonies were obtained from the plate and used to run the colony PCR. Also, using pNW33N-Gas6 plasmid extracted from overnight culture, the Gas6 coding sequence was amplified using Gas6 signal top and bottom primers. Gel electrophoresis was run to testify the samples. As shown in Figure 7 below, an expected band size of 2069 bp was obtained.
Fig.7 Electrophoresis results of single colonies after transformation.
Line1: 1kb DNA Ladder. Line 1-9: Single colonies amplified by Aox and Gas6 forward primers; Line10: Gas6 coding sequence amplified from pNW33N-Gas6.
Suicide System Construction
Plasmid Extraction and Verification
The plasmids of PUC57-sok, PUC57-hok, pET28a-TRP were extracted from glycerol stock after an overnight culture. A gel electrophoresis was run to verify these plasmids.
Fig. 8 Purity and concentration results of the extracted puc57+sok through spectrophotometer.
The sample of plasmid extracted in this experiment is sok. The OD260/280 ratio is 1.844, and the concentration of the sample is 562 μg/ml.
Fig. 9 Purity and concentration results of extracted puc57+hok using a spectrophotometer.
The sample of plasmid extracted in this experiment is puc57+hok. The OD260/280 ratio is 1.833, and the concentration of the sample is 880 μg/ml.
Fig. 10 Purity and concentration results of the extracted pET28A-TRP using a spectrophotometer.
The sample of plasmid extracted in this experiment is pET28A-TRP . The OD260/280 ratio is 1.879, and the concentration of the sample is 155 μg/ml.
Fig.11 Electrophoresis results.
Line1: 100bp DNA Ladder. Line2: Gas6 09/03. Line3: Gas6 09/03. Line4: Sok 09/03. Line5:Sok 09/29. Line6: LB+Zeocin E.coli TRP. Line7: Hok 09/01. Line8: P.pastoris Gas6. Line9: 1kb DNA Ladder.
Fig. 12 Electrophoresis results.
Line1: 1 kb DNA Ladder. Line2: hok. Line3: hok. Line4: hok. Line5: hok. Line6: sok. Line7: sok. Line8: sok. Line9: sok. Line10: sok. Line11: TRP1. Line12: TRP1. Line13: TRP1. Line 14: 0.1kb DNA Ladder.
Basen on the results of gel electrophoresis, the plasmids of PUC57-sok and PUC57-hok were purified successfully. However, the samples of pET28a-TRP plasmid didn’t have any band showing on the gel, indicating that the extracted DNA might not be target plasmid.
In order to testify to the result, TRP was amplified by different primers and assembled with sok and hok sequences separately after linearization using Gibson Assembly. According to the gel electrophoresis result in Figure 13, there existed a bold band on Line 11, which was not consistent with the simulated gel. None of the gel results were consistent with the simulated gel. Thus, it was clear that the suicide system for our plasmid was not constructed, and the gel result also indicated that there was something wrong with original TRP plasmid.
Fig. 13 Electrophoresis result.
Line1: 1 kb DNA Ladder. Line2: TRP. Line3: 1st. Line4: 2nd. Line5: sok1. Line6: sok2. Line7: sok3. Line8: Suicide+sok primer. Line9: Suicide+ hok primer. Line10: Suicide+suicide primer. Line11: hok1. Line12: hok2. Line13: hok3. Line 14: 0.1kb DNA Ladder.
EGF Transformation and Expression in Southwest Jiaotong University
First, the pNW33N-T7-EGF plasmid was ordered from Genscript Company, and the glycerol stock containing the plasmid was cultured overnight. After plasmid extraction, the samples were loaded to gel for verification.
Figure 14. OD value of pNW33N-EGF plasmid.
The figure shows the sample of plasmid extracted in this experiment, which is the pNW33N-EGF. The OD260/280 ratio is 1.841, and the concentration of the sample is 564.4 μg/ml.
Fig.15 Electrophoresis result.
Line 1: 4500 DNA ladder, Line 2:pNW33N-EGF-1 plasmid, Line3: pNW33N-EGF-2 plasmid
Results from this gel electrophoresis experiment were not valid. Without clear bands of standard ladder, the band size could not be determined. Therefore, we tried to do the plasmid extraction for one more time.
Figure 16. OD value of pNW33N-EGF plasmid.
The figure shows the sample of plasmid extracted in this experiment, which is the pNW33N-EGF. The OD260/280 ratio is 1.841, and the concentration of the sample is 295.7 μg/ml.
Fig.17 Electrophoresis result of the pNW33N-EGF plasmid.
In the gel, Lane 1: 5000 DNA ladder. Lane 2: pNW33N-EGF plasmid sample 1. Lane 3: pNW33N-EGF plasmid sample 2. Lane 4: pNW33N-EGF plasmid sample 3, Lane 5: pNW33N-EGF plasmid sample 5, and Lane 6: 1kb DNA ladder
Compared with the ladder, the sizes of EGF plasmids in bands 2 or 3 was as expected.
Plasmid Transformation
For plasmid transformation, electrotransformation was used to transform the above plasmid into E. coli BL21 (DE3) and incubated overnight on LB+CMR plates, but the single colonies were not obtained because the amount of antibiotics was not appropriate.
Figure 18. Transformation results of BL21 transformed with pNW33N-EGF.
Experimental group: pNW33N+EGF in BL21 (LB+CMR), Blank control: BL21 (LB+CMR) Conclusion: No target single colonies were generated in the experimental group.
Fig 20. PNW33N-EGF E.coli with CMR.
The figure depicts the transformed plasmid pNW33N+EGF in E. coli (LB+CMR). The clear plates indicate a lack of colonies, so the transformed colonies were not able to grow on these plates.
Fig 21. PNW33N-EGF E.coli without CMR.The figure here is depicting the transformed plasmid pNW33N+EGF in E.coli.
These plates are grown without CMR and target single colonies were generated on the plates.
In conclusion the bacteria grew more on the plate that contained no CM than the plate that contained CMR. Therefore, the antibiotic selection worked and further colony PCR is required for verification.
Gas6 Transformation and Expression in Southwest Jiaotong University:
The pPICZaA-Gas6 plasmid was ordered from Genscript Company, and the glycerol stock containing the plasmid was cultured overnight. After plasmid extraction, the samples were loaded to gel for verification.
Fig 22. OD value of PPICZαA-Gas6-3 plasmid.
The figure shows the sample of plasmid extracted in this experiment, which is the PPICZαA-Gas6-3. The OD 260/230 ratio is 2.070, and the concentration of the sample is 144.9 μg/ml.
Fig.23 Electrophoresis results of the PPICZαA-Gas6 plasmid.
Line1:5000 DNA Ladder, Line2:PPICZαA-Gas6-1, Line3:PPICZαA-gas6-2
Fig.24 Electrophoresis result of PPICZαA-Gas6 plasmid.
Line1:5000 DNA Ladder, Line2:PPICZαA-Gas6(PCR), Line3:PPICZαA-gas6-1
Compared with the marker, the band size of PPICZαA-Gas6 plasmid in the third lane was as expected. This sample was used to perform an electric transformation into Pichia pastoris GS115. Single colonies were selected for colony PCR after they grew on the plate with zeocin. Finally, electrophoresis verification was performed, but no valid results were obtained. Therefore, we decided to try chemical transformation for one more time.
Fig.25 Chemical transformation result of PPICZαA-Gas6 plasmid.
The chemical conversion was tried again, and the selective plate did not have any bacteria growing on it, indicating the failure of chemical transformation.
Gas6 Transformation and Expression in Georgia State University:
The pPICZaA-Gas6 plasmid was ordered from Genscript Company. After transforming the plasmid material into E.coli, more culturing was carried out to get more plasmid materials. After plasmid extraction, the samples were loaded to gel for verification.
Fig.26 Concentration and purity results of the extracted pPICZα-Gas6 through a spectrophotometer.
The sample of plasmid extracted in this experiment is pPICZα-Gas6. The OD 260/280 ratio is 1.833, and the concentration of the sample is 248μg/ml.
Fig.27 Electrophoresis results. Line1: 1 kb DNA Ladder. Line 2-5: TRP. Line 6-11: single colony of E.coli pPICZaA-Gas6. Line 12-13: pPICZaA-Gas6 extracted from E.coli. Line 14: 0.1kb DNA Ladder.
A band size around 2000bp was expected for the pPICZαA-Gas6 plasmid. Therefore, the samples in Lane 12 and 13 were used for electro-transformation.
Fig.28 Transformation results of Pichia Pastoris transformed with pPICZαA-Gas6.
Many colonies were obtained after overnight incubation.
The colonies we obtained indicated that the transformation of pPICZαA-Gas6 was successful. Single colonies were picked for overnight culture and the extraction of more plasmids.
Fig.29 The image is depicting the electrophoresis results of the different primers.
Line1: 1kb DNA Ladder. Line2: Single colony① on plate(Gas6 signal bottom primer+ Gas6 signal top primer). Line3: Single colony② on plate(Gas6 signal bottom primer+ Gas6 signal top primer). Line4: Single colony③ on plate(Gas6 signal bottom primer+ Gas6 signal top primer). Line5: Single colony④ on plate(Gas6 signal bottom primer+ Gas6 signal top primer). Line6: Single colony⑤ on plate(Gas6 signal bottom primer+ Gas6 signal top primer). Line7: Single colony⑥ on plate(Gas6 signal bottom primer+ Gas6 signal top primer). Line8: Single colony⑦ on plate(Gas6 signal bottom primer+ Gas6 signal top primer). Line9: Colony sample⑧ from solution(Gas6 signal bottom primer+ Gas6 signal top primer). Line10: Colony sample⑨ from solution(Gas6 signal bottom primer+ Gas6 signal top primer).
Fig.30 Purity and concentration results of Pichia Pastoris single colony transformed with pPICZαA-Gas6 through spectrophotometer.
The OD260/280 ratio is 1.979, and the concentration of the sample is 144 μg/mL.
Fig.31 Electrophoresis result.
Line1: 1kb DNA Ladder. Line2:pPICZαA-Gas6 E+P. Line3: pPICZαA-Gas6 EcrRⅠ. Line4:Gas6 09/01. Line5: pPICZαA-Gas6 E+P. Line6: pPICZαA-Gas6 EcrRⅠ. Line7: Gas6 09/03. Line8: Gas6 09/03. Line9:P. Pastoris Gas6. Line10: pPICZαA-Gas6 extracted from E.coli single colony. Line11: pPICZαA-Gas6 E+P. Line12: pPICZαA-Gas6 EcrRⅠ. Line13:pNW33N-T7-Gas6 09/29. Line14: 100bp DNA Ladder.
After verification of colony PCR and gel electrophoresis, two single colonies were cultured in solution media. The BMMY and BMGY medium were prepared and applied for inducing Gas6 expression in Pichia pastoris.
Recombinant Pichia pastoris and induced expression.
Fig.32 OD value of Pichia pastoris in BMMY medium at 600nm through spectrophotometer. The Pichia pastoris in BMMY medium showed an absorption reading of 0.034A.
Fig.33 OD value of Pichia pastoris in BMMY medium at 600nm through spectrophotometer. The Pichia pastoris in BMMY medium showed an absorption reading of 0.054A.
After protein expression, the supernatant and pellet were separated. Supernatant was concentrated and the pellet was lysed before SDS-PAGE began. SDS-PAGE result is shown in Figure 9. It was known that the size of gas6 is 75kDa. However, there existed no bands with 75 kDa in the SDS-PAGE result. Later, western blotting was used to testify the existence of Gas6 protein. The result of WB indicated that there was no Gas6 protein in any of our samples.
Fig.34. SDS-PAGE result.
Line 1: protein ladder. Line 2: empty. Line 3: glucose supernatant. Line 4: glucose cell extracts. Line 5: methanol supernatant. Line 6: methanol cell extracts. Line 7: glucose supernatant.
Fig.35. Western blotting result.
The first and last line were protein ladders and these showed observable visible bands on the gel. No bands were observed between the protein ladders.
Fig.36 Purity and concentration results extracted by a single E.coli colony transformed with pPICZαA-Gas6 through spectrophotometer.
The sample of plasmid extracted in this experiment is pPICZαA-Gas6. The OD 260/280 ratio is 1.955, and the concentration of the sample is 43.0 μg/mL.
Fig.37 Electrophoresis result.
Line1: 100bp DNA Ladder. Line2: E.coli single colony transformed with pPICZαA-Gas6. Line3: E.coli single colony transformed with pPICZαA-Gas6. Line4: E.coli single colony transformed with pPICZαA-Gas6. Line5: E.coli single colony transformed with pET28A-TRP. Line6: E.coli single colony transformed with pET28A-TRP. Line9: Pichia Pastoris single colony transformed with pPICZαA-Gas6. Line10: Pichia Pastoris single colony transformed with pPICZαA-Gas6. Line11: Pichia Pastoris single colony transformed with pPICZαA-Gas6. Line12: Pichia Pastoris single colony transformed with pPICZαA-Gas6. Line13: Pichia Pastoris single colony transformed with pPICZαA-Gas6. Line14: 1kb DNA Ladder.
In order to testify the transformation results again for analysis in our failure, plasmid was extracted from the colonies, and twelve more single colonies were used for conducting colony PCR. The band size was not as expected. No band around 2000bp was detected. The band size was more than 10000kp. Our plasmid material might have been contaminated during storage. In the future, we may need to do more research in our transformation methods, and DNA sequencing for all the existing materials is necessary.
