Part:BBa_K737031

Spot42 based small RNA1

Spot42 is a multitarget small RNA that mediates the discoordinate expression of the E.coli galactose operon and other sugar metabolic pathway,such as galK,nanC,ytfJ,srlA,which are the 5’ leader sequence of the corresponding metabolic enzymes.

Take galK for example,Spot42 causes translation repression by base pairing to RBS in the 5’ leader sequence,then block the recognition of the antiSD sequence on 30S subunit. It’s a very strong pairing(up to 20bases),but it only causes 2.6 fold repression according to Johannes H. Urban and Jo¨ rg Vogel,for it doesn’t result in the degradation of the RNA complex by RNaseE(ssRNA degradation) and RNaseIII(dsRNA degradation),both of them are major enzymes that causes RNA degradation in vivo.Spot42-galK complex degrades in a slow and presently unclear way. The base pairing is initiated by the recognition of seed region,which plays a significant role in the repression efficiency.

Small RNA chaperone Hfq is an abundant protein that has 40000~50000 copies each cell,accelerating annealing and strand-exchange between small RNAs and target mRNA.Hfq binds to the AU-rich domain of small RNA,and footprinting experiments has shown that Hfq protects the degradation of RNA from RNase.It is essential to most of small RNAs in E.coli,extremely enchances the repression efficiency.

Spot42 have a weak Hfq binding site(whereas galK weaker),an endogenous terminator(unclear efficiency,maybe weak),and the multitarget repression stem-loop.

ClaI site and SalI site are well-selected restriction sites,they are used to assembly the artificial region to the 5’ end of Spot42,are compatible for all of the Ampr and Chlr vectors(no unexpected restction sites),whereas all the Kanar vectors will be restricted into fragments when treated with SalI/ClaI.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Note

ClaI regcognizes ATCGAT,which can be blocked by GATC methylation,pay extra attention to this when you assembly artificial region to Spot42.

I put R0011(the simplfied lac promoter) in the upstream of the small RNA,so that its transcription rate can be controlled by the concentration gradient of the IPTG.