Denovo gene synthesis: The gluttonous snake strategy
Synthetic biology ideas can only be realized with various biological components. As iGEM Alumni, we often faced this situation in our previous iGEM journey: the advancement of wet experiments was often limited by the progress of target gene synthesis, and the speed of DNA fragment synthesis often became the limiting step of the project.
iGEM has accumulated a large number of Standard Parts over the past decade, which are open source for community use. iGEM's official Distribution Kit Plate has provided iGEM with a large number of gene originals. In order to solve the problem of gene synthesis, ONCE developed and tried a cheap and accurate ultra-fast de novo gene synthesis technology, and synthesized red myrrh alcohol synthase, GFP protein, MVA The technique has been used to synthesize several important parts such as MrBOS, ccBOS, GFP protein, MVA, etc.
Sometimes we need DNA fragments that are not very long. Occasionally these fragments can be obtained by PCR after extracting the genome of the target species, but more often these fragments cannot be obtained directly by PCR. Even if the genome of the target species can be extracted, there are often problems with intron inclusion and ethics. However, if whole gene synthesis is performed, it usually requires a delivery cycle of five days or more, as well as a high economic cost. At the same time, regional industrial development imbalances and political confrontations exacerbate this suffering.
At this time, performing denovo gene synthesis on your own becomes a good option. Ideally, the team can get the desired gene fragment as early as the morning of the third day of demand generation. That is, we can go from designed to a gene fragment of up to 1- 2 kb in length in less than 24 hours at the earliest."
When small fragments are synthesized using denovo genes, we have the option to ligate longer fragments by homologous recombination. Even fragments of several kilo bp can be obtained, perfectly suited for routine experiments and available immediately upon completion of the experiment, without additional delivery processes.
Our steps are outlined as follows.
- Obtain a target gene map containing the sequence at NCBI, Addgene or other sources.
- Design primers starting from the 5' or 3 end of the target gene at 59 bp each. the second primer starts at the 21st base of the first primer, the third primer starts at the 21st base of the second primer. And so on, except for the first primer, each primer starts at the 21st base of the previous primer, ensuring that the two adjacent primers have 20bp of overlapping sequence. In this way, the primers were designed to cover all the target gene sequences.
- every six adjacent primers are added as a set in 0.2 ml PCR octet tubes. The system we used is as follows: 10ul total system, 0.3ul (10uM) for each primer, 5.2ul DNA Polymerase and 3.6ul nucleic acid free water. pcr reaction annealing temperature is 55°C, 28cyc.
- 1ul of sample was added to DNA electrophoresis at the end of the reaction, and the success of each set of reactionswas detected by DNA electrophoresis on agarose gel.
- (optional step) using the Multi-Segment Homologous Recombination Kit, 10 fragments can be ligated per reaction. Each fragment has 257bp, and 2370bp of the target gene can be obtained in one reaction by homologous recombination. DNA recovery from agar gel after electrophoresis.
- (optional step) PCR reaction was performed by adding the target gene sequence obtained in step 5 using the first and most terminal primers of the target gene, and the resulting PCR product was sent for sequencing and identification after agar gel electrophoresis.
After more than fifty experiments in the whole process, we found that this strategy can maintain more than 95% success rate of experiments while synthesizing genes super fast and cheaply. We believe that this denovo gene synthesis strategy will help the iGEM community achieve ultra-fast and inexpensive synthesis of accessible biological components. We hope that the gluttonous snake strategy will help iGEM teams in underdeveloped regions and underfunded experiments to stop suffering from gene synthesis.
To help understanding, we show the primer design for the gluttonous snake strategy to synthesize MrBOS gene as follows:
Part
We characterized the usage of J23100 in cross-species. Expanded its usage.
A high FPP production strategy
Farnesyl diphosphate(FPP) is the precursor of terpenoid. While terpenoids are important bio-functional molecules, our strategy can help future teams who are working on terpenoids synthesis to achieve better production.
For details, see Results