During the gel purification of electrophoresis products, low purification efficiency often occurs, which can thus unfortunately lead to loss of the results of previous experiments. For this reason, we placed the empty adsorbent column in air to dry for about 5 minutes after the washing with buffer and placed the column and a clean centrifuge tube in a 55°C metal bath for 5 minutes after adding ddH2O before the elution step. This operations can ensure the DNA mostly dissolved in ddH2O, and added the eluate back to the adsorbent column again after elution and centrifuged again. By doing this, we can dramatically increase the efficiency of gel purification and thus obtain a higher concentration of DNA product.
When engineering our B.subtilis, we need to introduce 4 gene modules into the strain at the same time, but if we perform multiple transformations, it may lead to low transformation efficiency. We chose to modify the start codon and stop codon of each gene, add tags and ribosomal binding sites, and then link the three genes into a single segment and introduce them into a plasmid vector before transforming into the strain. By doing so, the expression of each gene can be ensured and the number of transformations can be reduced.
We hope this will provide an idea for other teams with the need of conjugating multiple parts.
In the experiment, we needed to ligate the four PCR fragments in the correct order before ligating them to the plasmid vector pHT01, and using the enzyme cleavage and ligation method would make the experimental process too tedious with too many non-target fragments that the final expression vector would be difficult to ligate correctly. Cloning Kit), but found that the four inserts could be successfully ligated, but it was difficult to ligate them to the linearized plasmid vector. For this reason, we have improved the process by first ligating the four fragments together using Overlap PCR and then using the Plus Multi One Step Cloning Kit to ligate them to a plasmid vector that has been linearized by enzymatic digestion. In this way, a large number of expression vectors can be efficiently obtained.
In doing electrotranformation, almost every protocols you can find are telling to keep 200rmp, 3h before streaking the bacteria from recovery medium onto the plates. However, in our practices, if you shake the bateria for 3 hours without observing it becoming turbid, you can definitely make it longer, for example 5-6 hours to make it turbid under your naked eyes. This help you to verify the bacteria is still alive and promot their proliferation in the LB broth. But not too long since there's no antibiotic in the recovery medium!
This year, we are managing the competition under a series of chanllenges, especially the suspension and interruptions caused by the Covid19 pandemic. Our university was closed in March and the suspension continued to the end of the semester. We were not allowed to enter our lab until the early July and the school shut down again for weeks just before the wiki freeze. So how to conduct our project under the effect caused by Covid 19 is quite a question not only for us as well as for the other iGEM teams.
Therefore, SJTU-BioX-Shanghai and Tongji-China have lanuched a activity among the 2022 iGEM teams, especially thoes in Shanghai who have experienced the city's suspension from April to June, to encourage them sharing expereinces of how to run and enjoy a successful iGEM competition under the unexpected pandemic. It is uncertain when the pandemic can be totally removed from our daily life, so we hope our expereiences and advice listed below shall help some future teams in the post-pandemic era.
Some problems we have disccused in the brochure:
How to conduct a brainstorm online just as effiency and joyful as offline? How to have a nice and fruitful talk with professors if you can't visit them by person? How to carry out human practice online?
SJTU-BioX-Shanghai and Tianjin write a little brochure to guide future teams as well as the publics to get to know DNA data storage briefly and thoroughly.
The little brochure is going through the history of DNA data storage as well as its development, advantages and drawbacks and some imaginations on how it can be used in the future.
click partnership to see more stories on our collaboration to make the little brochure.
click hereto find our brochure
We have improved the part of Dsup protein. Direct evolution processes have been conducted on Dsup, the major direction is to increase the binding affinity of Dsup to the DNA strands, and thus hope for a better protective function.
To see more details of the improvement of our part please visit Improvement of Part.