Julius B. Lucks/Antisense RNA Transcription Attenuation/Brantl Wagner Recapitulation

Goals

1. To verify that we can grow the plasmids given to us by Brantl.
2. To verify that we can re-create the transcriptional attenuation results (Table 2 of Sabine Brantl, E Gerhart H Wagner J Bacteriol (2002) vol. 184 (10) pp. 2740-7).
   1. in-vivo beta-galactosidase reaction
   2. RTPCR

Growing Plasmids

• Transform plasmids into Tg1 cells
• mini-prep
• test-digest
• sequence relevant regions
• If doesn't work in Tg1 cells, then try in DH10B

Verifying transcriptional and translational fusions work

• grow cells on plates that will trigger lacZ
  • start by making a constitutively active lacZ protein inside a cell and growing on a plate - maybe have a biobricked lacZ?
• should all have positive hits
• this will be a practice that we can do library screening with lacZ

Verifying Table 2

• do double transformations according to the combinations in Table 2
• when plate up, observe the look of colonies that have functional antisense gene
  • is the attenuation observable?
  • we might have to make constitutively active versions of pUCpIII and pMGI
• How do we perform the quantitative beta-galactosidase experiment?
  • do we get similar attenuation factors

RTPCR verification

• how do we go about doing this? Should we try to perform their equivalent of Figure 3? How do we do this with constitutively active anti-sense RNAs? Do we do a pulse of inducer, and then freeze transcription?
• this would be good to practice to measure how any library hits measure to the native version.