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Goal |
Utility |
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Create a platform for assembling and “booting up” a genome.
Some microbial species like E coli and B. subtilis are easy to edit – add, delete, rearrange genes. Most are not, either because they don’t take up DNA from the environment willingly or they grow so slow that even simply maintaining them in vitro is difficult.
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1. We currently have the methods to assemble large fragments of chemically synthesized DNA but no method for turning a piece of DNA into a functioning cell, aside from the method performed by the Venter Institute which only works on the smallest of bacterial chromosomes. With our strategy one could build the synthetic chromosome piecewise inside of Bacillus subtilis as part of the B subtilis chromosome. When the synthetic portion of the chromosome is complete it is segregated from the B subtilis chromosome and is now contained within a functioning cytoplasm without the need for transferring it to a competent recipient cell. This would allow for the construction of large chromosomes using modular parts or chemically synthesized gBlocks or larger assemblies.
2. Modifying or engineering a select few species of bacteria (E. coli, B. subtilis and a few others) is relatively simple and well-documented. But most species cannot be easily modified either due to the lack of transformation tools such as recombination plasmids or due to the complexities of their growth requirements. For example, the human pathogen Mycoplasm genetalium is very against transformation and even when transformation is possible, it grows very slowly so the time in between successive transformations is at least a week. If the Mycoplasm chromosome were to be hybridized within B subtilis, a scientist studying Mycoplasm could maintain a stock of these hybrid spores. They could rapidly make genetic changes to the Mycoplasm chromosome with B subtilis’s simple recombination protocols, then “boot” the Mycoplasm genome when ready.
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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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Strategy |
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Technical Challenges |
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For an initial proof of concept we are integrating the chromosome of Streptococcus thermophilus (the “guest”) into the chromosome of Bacillus subtilis (the “host”) creating a hybrid circular chromosome. S. thermophilus was chosen because it’s not human pathogenic, has a similar overall structure to B. subtilis, and much of the housekeeping machinery is very similar at the amino acid level while its DNA sequence is different enough from B. subtilis to avoid erroneous recombination during hybridization.
The guest chromosome will be inserted into a location within the host chromosome that we surrounded with lambda recombination sites (attR and attL). After confirming the successful hybridization of the chromosomes through sequencing, lambda recombinase will be expressed to homologously recombine the two att sites at the hybrid boundary to re-segregate the host and guest chromosomes. After cell division we assume some percent of cells will only have the (formerly) guest chromosome. Any cells containing the host chromosome will then be killed using an inducible toxin on the host chromosome.
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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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But will it blend? |
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If you build it they will come |
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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 was confirmed both by our B. Subtilis surviving in the presence of lactose and by gel electrophoresis.
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More petri dishes, as performed by the Blue Hand Group.
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Tomorrow is another day |
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Back to the future |
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Unfortunately, at this point, someone found a calendar and informed us we were out of time.
We still believe at some point soon we will have a Streptococcus thermophilus genome fused to a Bacilus subtilis genome, and we'll be able to use loxP sites to cut the DNA into two loops, a Bacilus genome and a Streptococcus genome. We hope some of these dual-genome bacteria reproduce, and that at some point divisions occur in which the daughter bacteria contain only the Streptococcus genome.
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And that's when you came up with the idea for the flux capacitor.
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