May
June
July
August
September
October
May
From May 1st to May 10th, we decided that the topic of iGEM was "Influenza virus displayed by neuraminidase targeting tumors with high expression of transferrin receptor 1", and designed the initial experimental plan.
From May 10th to May 15th, we attempted to construct the A6-Ins15 plasmid through homologous recombination. While becoming familiar with the procedure, our aim was to create the first plasmid. Unfortunately, all the results turned out to be false positives, indicating unsuccessful mutation. As a result, we redesigned the homologous recombination primers and designed primers for point mutation. We attempted to insert these 45 bases through point mutation. Simultaneously, we also designed the primers required for constructing the A6-Ins30 plasmid.
From May 15th to May 21st, we successfully reconstructed the A6-Ins15 plasmid using a combination of homologous recombination and point mutation. Additionally, we constructed the A6-Ins30 plasmid through homologous recombination. The sequencing results confirmed the successful insertion of all the sequences.
From May 22nd to May 31st, we extracted a small amount of plasmids from the successfully constructed A6-Ins15 and A6-Ins30 plasmids in preparation for future virus rescue experiments. Additionally, we designed primers for constructing A6-HAps and A6-NAps plasmids to exchange packaging signals. Plasmids requiring packaging signal exchange needed DNA fragments to be inserted at both the 5' and 3' ends, requiring two consecutive homologous recombination steps for successful construction. On May 30th, we obtained the results of the first round of testing, some of which indicated successful insertion of the 5' end packaging signal.
June
From June 1st to June 11th, we extracted plasmids from the previously tested samples and performed a second round of 3' end packaging signal replacement. On June 11th, we received the sequencing results, and some samples showed successful insertion of the 3'-end packaging signal. During this time, we carefully compared the sequences and discovered that we missed an initial codon in the NA packaging signal at the 5' end of the A4-NAps plasmid. Additionally, a base was inserted at the end of the HA packaging signal at the 5' end of the A6-HAps plasmid. To address these issues, we designed a point mutation primer to improve these parts of the plasmids.
From June 12th to June 15th, we successfully performed the point mutation and obtained the sequencing results on June 14th. All the samples achieved the desired point mutation. As a result, we successfully constructed the A4-NAps plasmid and A6-HAps plasmid. We also extracted a small amount of plasmids for subsequent virus rescue experiments. During this time, we attempted the rescue of wild-type, S15, and S30 influenza viruses for the first time. After 3 days, we collected the culture medium and removed cell debris through centrifugation. However, the harvested virus supernatant did not show hemagglutination inhibition of chicken red blood cells in the hemagglutination inhibition experiment, including the wild-type virus. This may be attributed to unskilled operation.
From June 15th to June 20th, we attempted to rescue the wild-type, S15, S30, and PS mutant viruses again. After 3 days, we collected the culture medium and removed cell debris through centrifugation. Unfortunately, the harvested virus supernatant still did not show hemagglutination inhibition of chicken red blood cells in the hemagglutination inhibition experiment (Figure 2). We suspected that the virus titer was too low. To investigate further, we directly used the virus supernatant to infect HEK293T cells. After 4 hours, we extracted RNA, performed reverse transcription into cDNA, and attempted to amplify the NA protein and HA protein genes through PCR to determine if the corresponding genes were obtained. Unfortunately, we were unable to amplify the NA protein and HA protein genes (Figure 3).
During the final period from June 20th to June 30th, the experimenters needed to prepare for exams and suspended the experiment.
July
From July 1st to July 12th, the experimenters needed to prepare for graduate student interviews and participate in the 10th iGEMer exchange meeting in CCiC China, and suspended the experiment.
From July 13th to July 28th, we repeated the operations of virus rescue, virus infection, RNA extraction, and PCR. We made constant adjustments to the virus infection time, annealing temperature, and PCR cycle times. During this period, we successfully detected the target band several times through PCR, but all the sequences turned out to be wild-type after being tested (Figure 4). Suspecting an issue with the plasmid, we performed plasmid analysis. Upon comparing the sequencing results, we found no obvious problems with the plasmid.
August
From July 29th to August 5th, the experimenter went out to participate in a social practice activity and suspended the experiment.
From August 6 to August 12, we repeated the previous processes: virus rescue, virus infection, RNA extraction, PCR, etc. However, we still did not obtain any positive results. Suspecting that the issue may lie with the design itself, I decided to make changes. For the neuraminidase extension, we found a new paper on length modification of neuraminidase based on the WSN strain. Based on this paper, we designed related primers for SD24, SD9, SI14, and SI28. As for the packaging signal exchange, we planned to carry out synonymous mutations on the remaining packaging signals in the coding region. However, due to the lack of specific instructions in the literature regarding the procedure for synonymous mutation, we temporarily put that on hold.
From August 12th to August 20th, we repeated the processes of virus rescue, virus infection, RNA extraction, and PCR for S15, S30, and PS mutant viruses. Fortunately, the sequencing results showed successful insertion for S15 (Figure 5). However, it did not effectively cause a cytopathic effect (CPE) in HEK293T cells (Figure 6). During this time, we also constructed the plasmids A6-SD24, A6-SD9, A6-SI14, and A6-SI28. The sequencing results confirmed the successful construction of these four plasmids.
From August 20th to August 31st, we conducted virus rescue experiments for SD24, SD9, SI14, and SI28. After harvesting the virus supernatant, we performed the hemagglutination inhibition experiment using chicken red blood cells. Unfortunately, the results showed that none of the viruses effectively inhibited the agglutination of chicken red blood cells (Figure 7). Nonetheless, we proceeded with the virus infection experiment, extracted RNA for testing, and observed the cytopathic effect (CPE) in cells. Fortunately, both SD9 and SI14 showed successful insertion in the sequencing results, and obvious CPE effects were observed in the cells (Figure 9). Consequently, we prepared a large quantity of plasmids for the twelve-plasmid system, including A6-SD9 and A6-SI14 plasmids, for the large-scale production of viruses. Meanwhile, we decided to discontinue the rescue attempts for the S15 and S30 viruses. However, we persisted in our efforts to rescue the PS mutant virus, but unfortunately, we did not achieve any positive results.
September
From September 1st to September 10th, we prepared a large quantity of wild-type influenza viruses along with SD9 and SI14 mutant viruses. We collected 100 ml of wild-type virus supernatant and 150 ml of SD9 and SI14 virus supernatant. Subsequently, we concentrated all three viruses to 2 ml using ultracentrifugation. The concentrated virus solution exhibited significant hemagglutination inhibition of chicken red blood cells (Figure 10). We then performed absolute quantification of these three viruses (Figure 11) and discovered that the wild-type virus had the lowest titer, possibly due to premature harvest. Additionally, after discussing with my colleague in the laboratory, we decided to carry out synonymous mutations on the remaining packaging signals in the coding regions of the A6-HAps and A4-NAps plasmids for the PS mutant virus. The specific method involved humanizing these sequences using a codon optimization website. We synthesized the corresponding gene fragments and designed corresponding primers, which were then sent to our company for synthesis.Moreover, we also tried to construct a cell model with high TfR1 expression on CHO cells by spreading it in a 12-well plate.Cells were added with 1 ml of 1ml 2%FBS DMEM medium. After 24 hours, 1 ug pCMV3-TfR1 plasmid was transfected, and 6 hours after transfection, 1 ml of 2% FBS DMEM medium was transposed. Two days later, the protein was extracted for Western blotting to see the expression of TfR1. The results showed that we successfully constructed CHO cells with high TFR1 expression (Figure 12).
From September 10th to September 20th, we successfully performed synonymous mutations on the residual packaging signals at the 5' and 3' ends of the coding regions of the HA protein and NA protein in the A6-HAps and A4-NAps plasmids. We constructed A6-HAps(mut) and A4-NAps(mut) plasmids and extracted a small amount of plasmids for the rescue of the PS(mut) mutant virus. In a 6-well plate, we successfully rescued SD24 and SI28 mutant influenza viruses. The results indicated that we successfully deleted 24 amino acids/inserted 28 amino acids at the gene level. Additionally, to provide characterization, we included agarose gel electrophoresis images of the PCR-derived genes (Figure 13). Following the success of SD24 and SI28, we promptly prepared a large quantity of A6-SD24 plasmids and A6-SI28 plasmids for virus preparation. Furthermore, we also evaluated the targeting of the previously prepared large quantities of SD9 and SI14. Due to the low titer of the wild-type influenza virus, our initial batch of large-scale prepared influenza viruses only underwent one time of targeting evaluation. The remaining small amount of virus was utilized in Western blotting experiments. By exploring different conditions, we determined the appropriate electrophoresis conditions and verified the shortening and lengthening of the neuraminidase protein at the protein level (Figure 14).
From September 21st to September 30th, we made extensive preparations for the second and third batches of viruses. The second batch included preparations of WT, SD9, SI14, and SI28, while the third batch consisted of SD24 and SI28. In the second batch of viruses, the titer of SI28 was low, and it was consumed after two targeted experiments. Therefore, the third batch of viruses was prepared together with SD24. After the preparation of the third batch of viruses, we collected all the influenza viruses with different lengths of neuraminidase that we designed. Consequently, we conducted an experiment to inhibit the agglutination of chicken red blood cells (Figure 15). The results revealed that SD24 and SI28 did not effectively inhibit the agglutination of chicken red blood cells, presumably due to their low virus titer. Consequently, we performed absolute quantification of these viruses and confirmed that the virus titers of SD24 and SI28 were indeed significantly lower than those of WT, SD9, and SI28. The low titer of SI28 contradicted the original literature and made us realize that there was an issue with our design. Upon investigation, we discovered that the wrong sequence had been inserted into our SI28, prompting us to redesign the related primers. Additionally, we conducted a small-scale test on PS(mut) in a 6-well plate, but unfortunately, it was not successful this time. Furthermore, we evaluated the targeting of a large number of prepared viruses. However, due to the low virus titer of SD24, we only evaluated the targeting of the other three mutant viruses and the wild-type viruses, excluding SD24.
The following figure shows the reverse view of the above figure, which is SI28, SI14, SD9, SD24 and WT from top to bottom.
October
From October 1st to October 10th, we summarized and analyzed the data from the previous targeted experiments (Figure 17). Simultaneously, we reconstructed the A6-SI28-new plasmid and conducted virus rescue experiments with two mutant viruses, SI28-new and PS. However, we were still unable to successfully rescue the PS mutant virus. Upon examining the A4-NAps(mut) plasmid, we discovered that the start codon remained within its 5' packaging signal. Consequently, we performed a point mutation on the plasmid.
