Coding

Part:BBa_K4515004

Designed by: Kong Yangyang   Group: iGEM22_East_China   (2022-08-22)
Revision as of 07:16, 26 September 2022 by Yxq0603 (Talk | contribs)

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thl-opt

thl-opt

Profile

Name: thl-opt

Base Pairs: 1179 bp

Origin: Clostridium acetylbutyrate ATCC824, genome

Properties: acetyl-CoA acetyltransferase

Usage and Biology

BBa_K4515004 is an enzyme named thl, which encodes acetyl-CoA acetyltransferase and converts Acetyl-CoA into Acetoacetyl-CoA in the N-butanol biosynthesis pathway [1-2]. It has been found that N-butanol can be synthesized naturally in Clostridium, but the tolerance of Clostridium bacteria is not good enough for large-scale production [3-5]. Recently, Lactobacillus Brevis with better N-butanol tolerance has been isolated by researchers. In order to improve the yield of N-butanol, we optimized the codon of the thl gene and inserted the optimized thl DNA fragment downstream of the Pthl promoter (Figure 1), and transformed it into L. brevis ATCC367 competent cells to complete the N-butanol metabolic pathway [6].

Figure 1. the plasmid map of the composite part BBa_K4515005.

Profile

Name: Pthl promoter

Base Pairs: 375 bp

Origin: Clostridium acetylbutyrate ATCC824, genome

Properties: promoter of the thl gene

Usage and Biology

Pthl promoter is a constitutive promoter amplified from the ATCC824 genome which is upstream of the gene thl and it regulates the expression of the downstream genes. In this project, we employed this promoter to regulate the transcription of the thl gene.

Reference

1.Li, J., Zhao, J. B., Zhao, M., Yang, Y. L., Jiang, W. H., & Yang, S. (2010). Screening and characterization of butanol-tolerant micro-organisms. Letters in applied microbiology, 50(4), 373–379. https://doi.org/10.1111/j.1472-765X.2010.02808.x

2.Berezina, O. V., Zakharova, N. V., Brandt, A., Yarotsky, S. V., Schwarz, W. H., & Zverlov, V. V. (2010). Reconstructing the clostridial n-butanol metabolic pathway in Lactobacillus brevis. Applied microbiology and biotechnology, 87(2), 635–646. https://doi.org/10.1007/s00253-010-2480-z

3.Inui M, Suda M, Kimura S, Yasuda K, Suzuki H, Toda H, Yamamoto S, Okino S, Suzuki N, Yukawa H (2008) Expression of Clostridium acetobutylicum butanol synthetic genes in Escherichia coli. Appl Microbiol Biot 77:1305–1316. https://doi.org/10.1007/s00253-007-1257-5

4.Mitchell WJ (1998) Physiology of carbohydrate to solvent conversion by Clostridia. In: Poole RK (ed) Advances in Microbial Physiology, vol 39. pp 31–130

5.Bowles LK, Ellefson WL (1985) Effects of butanol on Clostridium-acetobutylicum. Appl Environ Microb 50:1165–1170

6.Biswas I, Jha JK, Fromm N. (2008) Shuttle expression plasmids for genetic studies in Streptococcus mutans. Microbiology (Reading). Aug;154(Pt 8):2275-2282. doi: 10.1099/mic.0.2008/019265-0.

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]


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Parameters
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