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Project Description |
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The Plan |
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We are using Bacillus subtilis as a backbone to incorporate the entire
genome of Streptococcus thermophilus. Once incorporated, we will use the
cre/lox recombination system to remove the Bacillus subtilis genome so
only the Streptococcus thermophilus genome remains. This will demonstrate
the use of Bacillus subtilis as a useful chassis to boot up an artificial
genome.
As you may have guessed from the sly shift in tense from present to future, we
did not actually succeed in transferring the entire genome of Streptococcus thermophilus
into Bacillus subtilis -- yet. We have developed, and will describe, a method which
we believe will enable us to do so, and the results we have obtained for the early
steps in that method have been encouraging. We still believe it may be possible to
accomplish our original goal, and will continue to pursue it in the weeks and months
to come.
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And so it begins...
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Why S. Thermophilus and B. Subtilis? |
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The bacteria |
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We chose these model organisms for this project because they're both gram positive low G-C ratio Firmicutes.
Since we're attempting a genome transplant, the fact that they're evolutionary cousins should help.
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Designing the plasmids |
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Putting it together |
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We decided to use E. coli to replicate our plasmids, which meant that the plasmids would not only need an E. coli origin of replication, but two ampicillin resistance genes we could use for selection -- one that would work in E. coli, and one that would work in Bacillus subtilis. We used a 6-piece Gibson assembly designed on SnapGene to create our initial plasmids with these features and the Streptococcus thermophilus homologous sequences that would become the landing pad for the S. thermophilus genome.
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Keoni explains the next step in the process, something he would do again, and again, and again...
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Confirming the transfer |
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Something sweet...with poison in it |
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After our E. coli had obliged by growing us a bunch of baby plasmids, we transformed Bacillus by stuffing it full of those plasmids. But how could we be sure?
Easy. We cultured the Bacillus on media which contained ampicillin. Unless the Bacillus had acquired ampicillin resistance by accepting the plasmid which contained our landing pad, it wouldn't grow. We'd used a similar tactic previously, selecting for the E. coli which had accepted our plasmid. Our landing pad had been inserted successfully -- so far, so good!
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Tony prepares petri dishes with agar and ampicillin.
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References
iGEM teams are encouraged to record references you use during the course of your research. They should be posted somewhere on your wiki so that judges and other visitors can see how you though about your project and what works inspired you.
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You can use these subtopics to further explain your project
- Overall project summary
- Project Details
- Materials and Methods
- The Experiments
- Results
- Data analysis
- Conclusions
It's important for teams to describe all the creativity that goes into an iGEM project, along with all the great ideas your team will come up with over the course of your work.
It's also important to clearly describe your achievements so that judges will know what you tried to do and where you succeeded. Please write your project page such that what you achieved is easy to distinguish from what you attempted.
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