Thoughts makes difference – this is what the Monkey King always said when he encountered difficulties on the Journey to the West, and it is also the most important point in the engineering process. Read more to find out about our engineering cycles, and see how we grew from mini monkeys to another Great Sage Equaling to Heaven.
After we have built the engineered strain, we continued to feed insect and bioassays as planned. We consulted information on the culture of Spodoptera litura, and carried out biological tests in a dedicated insect room.
Prepare a small plastic box, use a pinhole to pierce the lid of the plastic box to make it breathable, put the artificial feed of S. litura (made by Keyun Company) in a 50mL syringe (note to remove the needle), squeeze artificial feed into each small plastic box, squeeze the feed about 1cm long each time and press it flat, use a pipette to absorb 200μL of bacterial liquid on the feed, open the lid and air dry it naturally for 30min~1h, and wait for the artificial feed to completely absorb the bacterial solution before the biological test can be carried out. The hatched larvae were placed in a small feed box (10 S. litura larvae were placed in each box), and the data were weighed on various days of S. litura, and samples were taken to record its growth.
When experimenting, we operate strictly according to the design conditions. We monitor the preservation conditions of the larvae to ensure the stability of environmental factors. The early stages of the experiment went well, but during the larval pupation stage, we encountered an unexpected challenge: an unusually high larval mortality rate, a phenomenon that was also common in the control group. Despite our best efforts to simulate ideal pupation conditions, larval mortality is still high. This led us to rule out the potential effects of Artificial microRNAs (amiRNAs), as the larvae were unable to normalize pupation even without introducing any exogenous agents.
We recognize that successful engineering requires a deep understanding of the mechanisms by which things break down or fail. During the experiment, we found that S. litura did not feed when pupating. After reviewing the literature, we found that we ignored the key factor that S. litura needs to pupate in sandy soils. Under natural conditions, S. litura nests and pupates in sandy soils, which our experimental environment does not provide. In this case, we do not fully understand the specific environmental needs of S. litura. This information suggests that we may need to rethink our feeding strategy.
In this cycle of experiments, we will replace the feed with sand in the rearing container to simulate the natural pupation environment of S. litura. We will also adjust the feeding density to reduce interfering with the larvae and optimize the feed formula to provide a more complete range of nutrition. With these improvements, we hope to be able to effectively reduce the mortality of larvae and successfully promote their pupation and emergence.
Increase the amount of feed appropriately in the early stage of insect rearing. The larvae of S. litura pupate around 25 days. The amount of larval feed is significantly reduced. At this point, sand will be used instead of feed in a rearing container to simulate the natural pupation environment of S. litura so that it can pupate smoothly.
During the experiment, the pupation of S. litura in the control group was normal, and the results of the amiRNA group showed pupation deformity, as shown in Figure 1. The effectiveness of the experiment was successfully verified.
Figure 1 S. litura feed on the following strains of bacteria: CK (empty vector pET28a control), VLP (VLP-expressing bacteria), amiRCHS1 (amiRCHS1-expressing bacteria), VLP-amiRCHS1 (VLP and amiRCHS1 co-expressing bacteria). Scale bar: 1 cm.
Through an iterative cycle of "design-build-test-learn", we learn from failure and improve the design of experiments. After learning the key information that S. litura needs sand to pupate, we adapted the protocol and successfully pupated the larvae and emerged into adults. This process not only enhances our understanding of the S. litura's life cycle, but also demonstrates the importance of learning from failure and continuous improvement in bioengineering.
Agrobacterium transformation is a method widely used in plant transgenics. It relies on the T-DNA region of Agrobacterium and can insert foreign genes into the chromosomal DNA of plant cells. However, this approach is primarily aimed at the transformation of the nuclear genome rather than the chloroplast genome.
Gene gunning is an effective method for chloroplast transformation. This method allows foreign DNA to pass through the chloroplast's double membrane and integrate into the chloroplas t genome. That's why we chose to use a gene gun for quality transliteration.
However, as undergraduates, we still wanted to verify whether Agrobacterium could perform plastid transformation in tobacco, so we carried out an experimental design.
We transformed the constructed plant expression vector into an Agrobacterium strain. Plant cells were infected with Agrobacterium, transformed plant cells were cultured, and positive plants were screened.
During the testing phase, we found that tobacco leaves transformed with Agrobacterium could not be grown on antibiotic-containing media, suggesting that Agrobacterium transformation is very inefficient or not feasible in terms of plastid transformation. This result is consistent with our expectation that Agrobacterium is not suitable for plastid transformation.
Figure 2 PCR was performed on the experimental leaves, but the results showed that the target gene was not detected.
Faced with the failure of the experiment, we conducted a thorough analysis. We revisited the data and confirmed that Agrobacterium transformation was indeed not suitable for plastid transformation, and that chloroplast transformation needed to take into account the high copy number of the chloroplast genome and the challenge of having foreign DNA pass through the inner and outer membrane barriers of chloroplasts. Gene gun conversion is a better option.
After confirming that Agrobacterium transformation was not suitable for plastid transformation, we decided to use gene gun technology for plastid transformation. Cut the transformed leaves into small pieces and screen on spectinomycin-containing RMOP medium to obtain a self-contained transfection system.
Tobacco plastid transformation was performed. Briefly, young leaves of aseptically grown tobacco plants were bombarded with DNA-coated 0.6 μm gold particles using a PDS-1000/He Biolistic Particle Delivery System (Bio-Rad). Bombarded leaves were cut into ~5 × 5-mm pieces and selected on RMOP medium containing 500 mg L−1 spectinomycin. Several independent transplastomic lines were obtained and subjected to two to three additional rounds of regeneration on spectinomycin-containing medium to select for homoplasmy.
Figure 3 Schematic diagram of gene gun bombardment.
Due to our lack of experience, we first tested the bombardment with pressure, and finally the 1100psi pressure bombardment was the best, so we chose 1100psi for the gene gun transformation. During the transformation process, different bombardment distances of 6 cm, 9 cm, and 12 cm were used for incubation and PCR, and the results are shown in Figure 4. Tobacco leaves with a bombardment distance of 9 cm grew better on bispecific antibody medium.
Figure 4 (A)Tobacco leaves with bombardment distances of 9 cm, 6 cm, 12 cm, and 12 cm grown on bispecific antibody medium. (B) Only the 1st and 5th showed the target band, i.e. the blade at a bombardment distance of 9 cm, was successfully transformed.
In the course of our test, we found that the bombardment distance of the gene gun has a very important effect on the chloroplast transformation effect, and the mass conversion test at different bombardment distances under the same pressure is significantly different.
From this cycle, we have a better understanding of the chloroplast transformation of the plasmid, which will guide us to the next cycle.