Before JCVI shutdown due to the COVID-19 pandemic, we were in the process of sending our mycoplasma transformed samples for sequencing. We were able to send 125 samples for sequencings. Of these, we had 1 successful strain, strain 3C10, 13 strains that had 1 successful strain so we have to pick additional ones to sequence, and the rest are in need of checking. In our second and third round of sequencing, we sent less samples (25uL instead of the previous 50uL) in a move to save reagents, but we found that this resulted in lower quality sequencing results. For these we have been examining the results to see if the issue is the sample itself or the sequencing results itself, and these samples will most likely need to be resent for sequencing. There is an additional 82 samples that have had been transformed into mycoplasma cells and just need to be prepared and sent for sequencing. There are also 71 samples that have been prepared correctly all the way up to the mycoplasma transformation step and just need to be transformed and grown up for a few days before being send for sequencing. This finally leaves 153 samples that are stuck in the preliminary steps of our protocol. Originally this meant either re-preparing the DNA from the E. Coli transformation or redoing the E. Coli transformation, but due to the bacteria samples sitting out the last 2 months we most likely will need to go back to the bacteria transformation step for all of these samples. We have had issues throughout the process with our first two plate's samples worth (96 samples) and almost that entire set is stuck in the redo E. coli transformation phase. This is most likely due to this plate experiencing evaporation during storage in the freezer, so we had less DNA accessible and thus resulted in lower transformation efficiency. Due to this, we might need outside assistance in re-preparing this plate through redoing the 3 PCR processes to create our engineered plasmid.

Page Leader: Melissa Amezola