Gene expression is a fundamental aspect in the construction
of
a minimal synthetic cell, and the use of chromosomes will be crucial
for the integration and regulation of complex modules. Expression
from chromosomes in vitro transcription and translation (IVTT) systems
presents limitations, as their large size and low concentration make
them far less suitable for standard IVTT reactions. Here, we addressed
these challenges by optimizing lysate-based IVTT systems at low template
concentrations. We then applied an active learning tool to adapt IVTT
to chromosomes as template DNA. Further insights into the dynamic
data set led us to adjust the previous protocol for chromosome isolation
and revealed unforeseen trends pointing at limiting transcription
kinetics in our system. The resulting IVTT conditions allowed a high
template DNA efficiency for the chromosomes. In conclusion, our system
shows a protein-to-chromosome ratio that moves closer to in vivo biology
and represents an advancement toward chromosome-based synthetic cells.