Composite

Part:BBa_K4907133

Designed by: Jincheng Lv   Group: iGEM23_XMU-China   (2023-09-16)
Revision as of 15:17, 11 October 2023 by Ysl (Talk | contribs)


I0500-B0034-cpn10-B0034-cpn60-B0015

Biology

cpn10

The chaperonins are 'helper' molecules required for correct folding and subsequent assembly of some proteins. These are required for normal cell growth, and are stress-induced. Chaperonin-60 (Cpn60) and chaperonin-10 (Cpn10) are essential proteins involved in ATP-dependent folding of several intracellular proteins in the bacterial cell. Folding of the nascent substrate polypeptide takes place in the large central cavity formed by each ring of the tetradecameric Cpn60. This large cavity is closed upon capping by the heptameric Cpn10 (1). Ferrer and colleagues' research revealed that the two chaperonins exhibit robust protein refolding activity at temperatures ranging from 4 to 12℃ in vitro. They also suggested the potential for enhancing the cold stress resistance of bacteria by introducing cpn10 and cpn60 genes into Escherichia coli (2).

cpn60

Cpn60 is vital for proper folding and assembly of intracellular proteins in bacterial cells, with its large central cavity being capped by Cpn10. This process is essential for normal cell growth and can improve cold stress resistance when cpn60 and cpn10 genes are introduced into Escherichia coli.

Usage and design

To expand the growth temperature range of our engineered bacteria, we have chosen to overexpress the partner proteins, Cpn10 and Cpn60, to enhance their cold tolerance. We used both BBa_I0500 and BBa_B0034 to construct the expression circuit and obtained the composite part BBa_K4907133 (Fig. 1), which was assembled into the vector pSB1C3 by standard BioBrick assembly. The constructed plasmid was transformed into E. coli BL21(DE3), then the positive transformants were selected by chloramphenicol and confirmed by colony PCR and sequencing.


Fig. 1 Gene circuits of BBa_K4907133.


Characterization

Agarose gel electrophoresis (AGE)

When constructing this circuit, colony PCR and gene sequencing were used to verify that the transformants were correct. Target bands (3656 bp) can be observed at the position between 3000 bp and 5000 bp (Fig. 2).

Fig. 2 DNA gel electrophoresis of the colony PCR products of BBa_K4907133_pSB1C3.

???

We constructed I0500-B0034-cpn10-B0034-cpn60-B0015_pSB1C3 (BBa_K4907133_pSB1C3, as the experimental group) and I0500_pSB1C3 (as the control group). We characterized its growth at 4 ℃. As shown in the figure, strains carrying cnp10 and cpn60 did not show better stress resistance at low temperatures. Due to time limit, we are unable to characterize it in more detail.


Fig. 3 ???

Reference

1. M. Ferrer, T. N. Chernikova, M. M. Yakimov, P. N. Golyshin, K. N. Timmis, Chaperonins govern growth of Escherichia coli at low temperatures. Nat Biotechnol 21, 1266-1267 (2003). 2. B. Taneja, S. C. Mande, Structure of Mycobacterium tuberculosis chaperonin-10 at 3.5 A resolution. Acta Crystallogr D Biol Crystallogr 58, 260-266 (2002).

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1205
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 2426
    Illegal BamHI site found at 1144
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 979
    Illegal AgeI site found at 1369
    Illegal AgeI site found at 2442
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 961
    Illegal SapI.rc site found at 1868


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