Chronic lung infections are a feature of cystic fibrosis (CF) that many patients experience even with the advent of highly effective modulator therapies. Identifying factors that impact in the CF lung could yield novel strategies to eradicate infection or otherwise improve outcomes. To complement published studies using laboratory models or RNA isolated from sputum, we analyzed transcripts of strain PAO1 after incubation in sputum from different CF donors prior to RNA extraction. We compared PAO1 gene expression in this "spike-in" sputum model to that for grown in synthetic cystic fibrosis sputum medium to determine key genes, which are among the most differentially expressed or most highly expressed. Using the key genes, gene sets with correlated expression were determined using the gene expression analysis tool eADAGE. Gene sets were used to analyze the activity of specific pathways in grown in sputum from different individuals. Gene sets that we found to be more active in sputum showed similar activation in published data that included RNA isolated from sputum relative to corresponding reference cultures. In the samples, had increased levels of genes related to zinc and iron acquisition which were suppressed by metal amendment of sputum. We also found a significant correlation between expression of the H1-type VI secretion system and CFTR corrector use by the sputum donor. An sputum model or synthetic sputum medium formulation that imposes metal restriction may enhance future CF-related studies.IMPORTANCEIdentifying the gene expression programs used by to colonize the lungs of people with cystic fibrosis (CF) will illuminate new therapeutic strategies. To capture these transcriptional programs, we cultured the common laboratory strain PAO1 in expectorated sputum from CF patient donors. Through bioinformatic analysis, we defined sets of genes that are more transcriptionally active in real CF sputum compared to a synthetic cystic fibrosis sputum medium. Many of the most differentially active gene sets contained genes related to metal acquisition, suggesting that these gene sets play an active role in scavenging for metals in the CF lung environment which may be inadequately represented in some models. Future studies of transcript abundance in CF may benefit from the use of an expectorated sputum model or media supplemented with factors that induce metal restriction.