Unnatural base pair compositions and methods of use
Disclosed herein are methods, cells, engineered microorganisms, and kits for increasing the production of polypeptides comprising one or more unnatural amino acids. Further provided are cells, engineered microorganisms, and kits for increasing the retention of unnatural nucleic acids encoding the unnatural amino acids in an engineered cell, or semi-synthetic organism.
1. An engineered host cell comprising:
a) a first nucleic acid molecule comprising an unnatural nucleotide;
b) a second nucleic acid molecule comprising:
i) a deletion of a gene encoding a transposition-associated protein or transposable element, wherein the transposition-associated protein comprises InsB and/or InsA, wherein the transposable element comprises IS1, wherein the deletion is an N-terminal deletion, a C-terminal deletion, a truncation at both termini, an internal deletion, and/or a deletion of the entire gene; or
ii) a gene encoding a mutated transposition-associated protein or transposable element, wherein the transposition-associated protein comprises InsB and/or InsA, wherein the transposable element comprises IS1, wherein the mutated transposition-associated protein or transposable element is not expressed, or is expressed at reduced levels; and
c) a third nucleic acid molecule encoding a modified nucleoside triphosphate transporter that imports unnatural nucleic acids into the cell, wherein the modified nucleoside triphosphate transporter comprises a nucleoside triphosphate transporter from Phaeodactylum tricornutum and further comprises an N-terminal truncation, a C-terminal truncation, or a truncation of both termini, wherein the third nucleic acid molecule is incorporated in a genomic sequence of the engineered host cell, or comprises a plasmid encoding the modified nucleoside triphosphate transporter.
2. The engineered host cell of claim 1 , wherein the nucleoside triphosphate transporter exhibits increased stability of expression in the engineered host cell relative to expression in an equivalent engineered host cell that does not comprise the second nucleic acid molecule.
3. The engineered host cell of claim 1 , wherein the nucleoside triphosphate transporter comprises PtNTT2.
4. The engineered host cell of claim 3 , wherein the PtNTT2 is under the control of a promoter selected from a pSC plasmid or a promoter from a lac operon.
5. The engineered host cell of claim 1 , further comprising:
a) a Cas9 polypeptide or variants thereof, and
b) a single guide RNA (sgRNA) comprising a crRNA-tracrRNA scaffold, wherein the combination of the Cas9 polypeptide or variants thereof and the sgRNA modulates replication of the first nucleic acid molecule comprising the unnatural nucleotide.
6. The engineered host cell of claim 1 , wherein the engineered host cell is a prokaryotic cell comprising an Escherichia coli cell or an Escherichia coli BL21 (DE3) cell.
7. The engineered host cell of claim 1 , wherein the unnatural nucleotide comprises an unnatural base selected from the group consisting of:
8. The engineered host cell of claim 1 , wherein the second nucleic acid molecule comprises a deletion of a gene encoding a transposition-associated protein, wherein the transposition-associated protein comprises InsB, or the second nucleic acid comprises a gene encoding a mutated transposition-associated protein, wherein the transposition-associated protein comprises InsB.
9. The engineered host cell of claim 1 , wherein the second nucleic acid molecule comprises a deletion of a gene encoding a transposable element, wherein the transposable element comprises IS1.
10. The engineered host cell of claim 1 , wherein the second nucleic acid molecule comprises a deletion of a gene encoding a transposition-associated protein, wherein the transposition-associated protein comprises InsB-4, and wherein the deletion comprises an N-terminal deletion, a C-terminal deletion, a truncation at both termini, an internal deletion, and/or a deletion of the entire gene.
11. The engineered host cell of claim 1 , further comprising a derepressed polB gene.
12. The engineered host cell of claim 1 , wherein the engineered host cell constitutively expresses or overexpresses DNA polymerase II.
13. The engineered host cell of claim 1 , wherein the transposition-associated protein comprises InsB-4 and/or InsA-4.
14. The engineered host cell of claim 1 , further comprising a deletion of the gene encoding RecA.