Identify replication origins in archaea with Chip-Seq

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      Lisa
      Participant

      Hey guys,
      we are currently working on a group project where we need to establish a workflow to proof the existence of origins of replication in an archaeal strain (N. maritimus) in vivo. So this question might be a very stupid question but unfoturnatly we haven’t been able to overcome it.
      So we’ve been thinking about doing Chip-Seq, we would grow the Strain in Batch cultures, express the recombinant Orc1/ Cdc6 proteins with hexa-his tag in E. Coli, purify, add them to the N.maritimus cells and then do chromatin immunoprecipitation followed by illumina sequencing.
      But we can’t think of a way to get rid of the native proteins, or how could we make sure that just the tagged proteins bind to the origins of replication? We’ve been thinking of knockout mutations, but as at least to our knowledge there is not much known about this strain or it hasn’t been genetically modified yet, we are not sure if this would be possible or how we could do the knouckout.
      Also regarding the his tag, how to decide wether to introduce the tag c- or n-terminal?
      Thanks 🙂

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