Part:BBa_K541501

Promoter Pveg, RBS spoVG and SacB signal peptide for B. subtilis

Constitutive promoter veg(BBa_K143012) coupled to the strong Ribosome Binding Site spoVG(BBa_K143021) from B. subtilis. These parts have been taken from 2008_Imperial_Collage. Pveg is a constitutive promoter that constitutively expresses the P43 protein in B. subtilis. SpoVG is an endogenous ribosome binding site from B. subtilis. The sequence of the spoVG ribosome binding site is AAAGGUGGUGA which is complementary to the sequence UUUCCUCCACU from the 3' region of the 16s rRNA from B. subtilis. SacB is a signal peptide used in the Sec-SRP (secretory signal recognition particle) pathway by B. subtilis. Signal peptides are responsible for directing preproteins (secretory proteins with a signal peptide region attached) through an appropriate secretory pathway. In the case of the Sec-SRP signal peptide, they direct preproteins from the cytoplasm into the growth medium.

Note: According to our results, our SacB bound LALF expressed into supernatant which could inhibit gram negative bacteria E.coli growth by interacting with LPS layer. That is, the part can be used into extracellular expression.

Experiment of BBa_K541515

We aimed to stop E.coli growth by binding LPS layer on its cell Wall with our LALF protein. We performed several experiments whether the protein works or not. We spread E.coli with RFP onto four plates that two of them as control group and the other as experimental group. Additionally, we added 10 ul supernatant of B.subtilis liquid cultures onto one expermental plate and 10 ul supernatant onto the other one. Respectively, we saw that on the both of the zones where supernatant with LALF had been added, there was no E.coli colony.

We deduced that our protein works and stops E.coli growth both in liquid culture or supernatant.

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]

Functional Parameters: Austin_UTexas

Burden Imposed by this Part:

Burden Value: 14.4 ± 3.6%

Burden is the percent reduction in the growth rate of E. coli cells transformed with a plasmid containing this BioBrick (± values are 95% confidence limits). This part exhibited a significant burden. Users should be aware that BioBricks with a burden of >20-30% may be susceptible to mutating to become less functional or nonfunctional as an evolutionary consequence of this fitness cost. This risk increases as they used for more bacterial cell divisions or in larger cultures. Users should be especially careful when combining multiple burdensome parts, as plasmids with a total burden of >40% are expected to mutate so quickly that they become unclonable. Refer to any one of the BBa_K3174002 - BBa_K3174007 pages for more information on the methods and other conclusions from a large-scale measurement project conducted by the 2019 Austin_UTexas team.

This functional parameter was added by the 2020 Austin_UTexas team.