The blueprint for all life forms on Earth is written in a code consisting of four "letters": A, T, C and G, which pair up in the DNA double helix.
But the lab organism has been modified to use an additional two, giving it a genetic code of six letters.
Researchers hope the work could lead to bugs that can help manufacture new classes of drugs to treat disease.
The team from the US, China and France have published their work in PNAS journal.
Previous research had shown that an "unnatural base pair" (UBP), consisting of two synthetic letters called X and Y, could be incorporated into the DNA of Escherichia coli bacteria.
But the resulting bugs grew slowly, and the UBP was expunged after several rounds of cell division.
Now, Prof Floyd Romesberg, from The Scripps Research Institute in La Jolla, California, and colleagues, have shown that their single-celled organism can hold on indefinitely to the synthetic base pair as it divides.
"We've made this semisynthetic organism more life-like," said Prof Romesberg, senior author of the new study.
"Your genome isn't just stable for a day," said Prof Romesberg. "Your genome has to be stable for the scale of your lifetime. If the semisynthetic organism is going to really be an organism, it has to be able to stably maintain that information."
Key to the advance was a modification to a molecular transporter, which helps the E. coli bugs import the UBP.
Next, the researchers optimised their previous version of Y so that it could be better recognised by the enzymes that synthesise DNA molecules during replication.
Finally, the researchers set up a "spell check" system for the organism using the CRISPR-Cas9 genome editing tool.
They were able to take advantage of the tool to ensure that any cells that dropped X and Y would be marked for destruction.
Their semisynthetic organism was thus able to keep X and Y in its genome after dividing 60 times, leading the researchers to believe it can hold on to the base pair indefinitely.
"We can now get the light of life to stay on," said Prof Romesberg.
"That suggests that all of life's processes can be subject to manipulation."
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