Team:LA Biohackers/Project

Project Description

The Plan

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.

And so it begins...

Why S. Thermophilus and B. Subtilis?

The bacteria

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.

Designing the plasmids

Putting it together

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.

Keoni explains the next step in the process, something he would do again, and again, and again...

Confirming the transfer

Something sweet...with poison in it

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!

Tony prepares petri dishes with agar and ampicillin.

But will it blend?

If you build it they will come

So now we've confirmed that our landing pad has been built, but can our genome actually land there? At this point we went off-plan a little bit. Our goal was still to insert S. thermophilis, but while we were working out exactly how we would do that with the whole genome, we occupied ourselves with "proof of concept" experiments. This involved using yeast as a stunt double while our star thermophilus was still in make-up.

For this, we relied on positive selection, with a toxin that would be expressed in the presence of lactose. The gene containing the toxin would be replaced by the yeast sequence if all went well. We successfully inserted 5 kilobase, 20 kilobase, and 100 kilobase strands of yeast DNA using homologous sequences on our landing site, which were confirmed both by our B. Subtilis surviving in the presence of lactose and by gel electrophoresis.

More petri dishes, as performed by the Blue Hand Group.

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1. Overall project summary
2. Project Details
3. Materials and Methods
4. The Experiments
5. Results
6. Data analysis
7. Conclusions

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