Biology

Ligation of rposLS amplicons into PSB1C3 and transformation. Screen by PCR, restriction of four clones and sequencing. Construction with fhaLS from pTOPO (EcoRI and PstI) and I13401 (XbaI and EcoRI) and construction with rsmA PCR products. R0011-rsmA-pSB1A2 (construction 31).With R0011 digested by SpeI and PstI and R0011digested by XbaI and PstI.

fhaSL-I13401-pSB1AT2 (construction 32) Four positives clones per construction were selected after colony PCR and sent for sequencing. (No restriction screening was performed in order to gain times)

Biology

Sequencing result of construction 29, 30, 31 and 32 are positives. Glycerol stock was performed. Construction from previews construct.

• R0040-fhaLS-I13401-pSB1A2 (construction 35 : 31 digested by XhoI and PstI and R0040 digested by SalI and PstI)
• R0040-fhaLS-I13401-pSB6A1 (construction 34: 3A assembly with 31 digested by XbaI and PstI, R0040 digested by EcoRI and SpeI and pSB6A1 digested by EcoRI and PstI)
• R0011-rsmA-pSB3C5 (construction 33: clone 31 and pSB3C5 digested by EcoRI and PstI)

The colony PCR screen was performed for construction 33 and 35. One clone for construction 35 and four clones for construction 33 were sent for sequencing. Only one clone for construction 35 appeared fluorescent using the epi-fluorescent microscope visualization. Sequencing was correct for construction 33 and 35

Double transformation was performed for rsmA inhibition tests with construction 33 and 35. Flow Cytometry of K253006, constructs 30 and 35 in order to measure the strength of fha and mag. Flow Cytometry of double transformants (33 and 35) in order to measure the effect of rsmA (33) on the translation of GFP mRNA.

Test of RBS construct (K256003, 30, 35 and double transformed cells contained construct 30 and 35 with or without IPTG) by FACS and Fusion.