Team:Shanghai United/Results
Results
First Attempt to Construct the Plasmids
AmilGFP PCR
Figure 1: Gel Electrophoresis Results of PCR of amilGFP Genes
Figure 1 shows gel electrophoresis results of amilGFP PCR. Column M is a 2K marker ladder. Columns 1-6 are PCR products of amilGFP genes
All 1-6 columns displayed successful results at 700bp which could be used for later experiments.
ARS Plasmids Extraction
|
|
Group 1 |
Group 2 |
|
ARSA |
425.9 ng/μL |
307.8 ng/μL |
|
ARSD |
293.4 ng/μL |
300.8 ng/μL |
|
ARSR |
214.4 ng/μL |
154.0 ng/μL |
Table 1: Concentrations of ARS Plasmids Extraction Results
Table 1 shows that all concentrations except the concentration of ARSR from Group 2 are within the desired range. While Group 2 was collecting ARSR, half of the sample was accidentally mixed with ARSA samples and must be discarded - hence the deviation. The concentrations were used in to calculate the amount of ARS genes to be added in restriction enzyme digestion.
AmilGFP PCR Products Clean-up
|
|
Group 1 |
Group 2 |
|
amilGFP |
382.6 ng/μL |
350 ng/μL |
Table 2: Concentrations of amilGFP PCR Products Clean-up Results
Table 2 shows the concentrations of products of amilGFP PCR clean-up. The concentrations of the samples are optimal and could be used to calculate the amount of samples added when performing restriction enzyme digestion.
ARS/amilGFP Digestion
Figure 2: Gel Electrophoresis Results of Restriction Enzyme Digestion of ARS Plasmids and amilGFP Genes
Figure 2 shows gel electrophoresis results of gel electrophoresis on restriction enzyme digestion products of ARSA, ARSD, ARSR, and amilGFP genes. All products showed successful results that could be used for later experiments and these target genetic materials were extracted from the desired bands.
ARS/amilGFP Gel Extraction
|
|
Group 1 |
Group 2 |
|
ARSA |
31.0 ng/μL |
28.1 ng/μL |
|
ARSD |
48.3 ng/μL |
23.3 ng/μL |
|
ARSR |
5.2 ng/μL |
20.5 ng/μL |
|
amilGFP |
106 ng/μL |
|
Table 3: the Concentrations of 7.30 ARS/amilGFP Gel Extraction from Two Groups
We recycled GFP and the three plasmids ARSA, ARSD and ARSR after electrophoresis. We used Nanodrop, a common lab spectrophotometer, to test the concentration of the four products. According to Table 3, all data except the concentration of ARSR from Group 1 are within the acceptable range. A possible reason for the anomaly is that when the sample containing ARSA was loaded into the gel box, the pipette pierced into the gel, causing a small amount of leakage of the sample. It is noted that the concentration of amilGFP is also abnormally high. The reason is that since the two samples of amilGFP used by the two groups are the same, we mixed them together and then tested theri concentration.
After we found out the concentration of amilGFP, ARSA, ARSD and ARSR, it will be much easier to calculate the volume of amilGFP needed to mix with each plasmid to construct T4 DNA ligase ligation system according to the ratio between amilGFP and plasmid is 1:10.
Colony PCR
Figure 3: Gel Electrophoresis Results of Colony PCR of First Experiment Attempt
We examined growths of ARS/amilGFP E. coli, cultivated since Jul. 30. Results were suboptimal: only 4 colonies were found in a total of 6 plates. We then performed colony PCR and gel electrophoresis to test for ARS/amilGFP genes in observed E. coli colonies. Figure 3 indicates that only one colony resulted to be ARSD/amilGFP positive, indicating failure of the experimental procedure.
Second Attempt to Construct the Plasmids
AmilGFP Digestion
Figure 4: Gel Electrophoresis Results of amilGFP PCR in Attempt 2
We performed PCR of the amilGFP gene to prepare for the second retry of the experimental procedure, then we performed electrophoresis to test for amilGFP PCR products. Figure 4 indicates that the second sample of group 1 resulted in failure, but the other samples are successful which could be retrieved for the later experiments use.
AmilGFP Gel Extraction
|
amilGFP |
92.3 ng/μL |
Table 4: Concentrations of Gel Extraction of amilGFP Enzyme Digestion Products
Table 4 showed the concentration of all products, extracted from gel from amilGFP Digestion, combined. The result is optimal and could be used to calculate the amount of sample added when performing T4 DNA Ligase Ligation.
Colony PCR
Figure 5: Gel Electrophoresis Results 1 of Colony PCR of Second Experimental Attempt
Figure 6: Gel Electrophoresis Results 2 of Colony PCR of Second Experimental Attempt
Figures 5 and 6 show the result for colony PCR identification on the E.coli with ARS/amilGFP inserted that were cultivated previously. The purpose is to examine whether the E.coli contains expected gene segment of ARSA/D/R and amilGFP.
As showing above, all samples are determined to have bands of corresponding molecular sizes, which shows that all selected colonies yielded positive results of ARS and amilGFP genes.
Function Tests
Function Test With NaH2PO4, Attempt 1
Such function tests were performed under the hypothesis that NaH2PO4 is structurally similar to arsenic compounds that were meant to be detected. NaH2PO4 is used to substitute for arsenic compounds due to experimental safety. No fluorescence was detected in all samples, indicating that the concentrations of NaH2PO4 may be too low or that genetically engineered E. coli will not react to NaH2PO4.
Function Test With NaH2PO4, Attempt 2
In order to verify our assumption in attempt 1, NaH2PO4 was used again in this testing, with greater concentration than the last test. No fluorescence was detected in all samples, indicating the inability of the genetically engineered E. coli to react with NaH2PO4.
Function Test With C2H6AsNaO2
Figure 7: Fluorescence Intensity of Transformed E. coli In Different Concentrations of C2H12AsNaO5 Solutions
Given that NaH2PO4 cannot be used to stimulate the expression of amilGFP in genetically engineered E. coli, C2H6AsNaO5 was used for this function test. Extremely minor fluorescence was detected by a microplate reader after 19 hours of reaction time. This result confirms the design of E. coli to be correct and functional.
Figure 7 displays the fluorescence intensity generated by an ARSD/amilGFP transformed E. coli reacting for 1 hour in C2H6AsNaO5 solutions. As seen in the figure, fluorescence was detected for all C2H6AsNaO5 concentrations that are above 0, which confirms that the designed plasmid worked as intended. However the fluorescence was rather minor, so we speculate that E. coli responds poorly to organic arsenic compounds. Further experiments would be conducted to test for fluorescence intensities of E. coli in inorganic arsenic solutions.
To be continued ...
