Biotechnology Bulletin ›› 2024, Vol. 40 ›› Issue (6): 310-318.doi: 10.13560/j.cnki.biotech.bull.1985.2023-1225
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LI Meng-ran1,2(), YE Wei2, LI Sai-ni2, ZHANG Wei-yang2, LI Jian-jun1(), ZHANG Wei-min2()
Received:
2023-12-29
Online:
2024-06-26
Published:
2024-06-24
Contact:
LI Jian-jun, ZHANG Wei-min
E-mail:1871288385@qq.com;lijianjun1672@163.com;wmzhang@gdim.cn
LI Meng-ran, YE Wei, LI Sai-ni, ZHANG Wei-yang, LI Jian-jun, ZHANG Wei-min. Expression of Lithocarols Biosynthesis Gene litI and Functional Analysis of Its Promoter[J]. Biotechnology Bulletin, 2024, 40(6): 310-318.
Fig. 2 PCR verification of gene litI A: Cloning of gene litI, M: DL5000 DNA marker; I: gene litI. B: PCR verification of bacterial liquid, M: DL5000 DNA marke; I1-5: positive colonies of pET-30a-litI
Fig. 3 Preliminary purification of protein LitI A: Optimization of protein LitI expression conditions. M: Blue Plus II protein marker(14-100 kD); 1, 3, 5 and 6: supernatant for ①, ③, ② and ④; 2, 4, 7 and 8: sediment for ①, ③, ② and ④. B: Preliminary purification of protein LitI, M: protein marker; 1: binding buffer; 2: 10% imidazole eluent
Fig.4 Bioinformatics analysis of LitI protein A: Hydrophilicity/hydrophobicity analysis of protein LitI. B: Secondary structure analysis of protein LitI. C: Homology modeling for tertiary structure prediction of protein LitI
Fig. 5 Construction of pGL3-basic-promoter recombinant vector and analysis of its transcriptional activity A: Cloning of the litI promoter fragments; M: DL5000 DNA marker; I-1, I-2: promoter fragments amplified by PCR. B: PCR validation of colonies with pGL3-basic-litI promoter; M: DL5000 DNA marker; 1-5: PCR validation of colonies containing I-1 promoter; 6-10: PCR validation of colonies containing I-2 promoter. C: Luciferase RU values of the pGL3-basic-unloaded/pgpd/litI promoter vector in Trans5α cells
Fig.6 PCR verification of Trans5α colony and promoter vector map A: litI promoter fragment amplified with -pET28a-Amp homology arm primers; M1: Trans2K DNA marker. B: Colony PCR of Trans5α-pET28a-I-2; M2: DL5000 DNA marker; C: vector map of Trans5α-pET28a-I-2-AmpR
Fig.7 Growth of E. coli containing promoter fragments on ampicillin-resistant plates A: The plates of Trans5α-pEASY-T1-AmpR, Trans5α-pET28a-ACP-AmpR, and Trans5α-pET28a-I-2-AmpR, P: Trans5α-pEASY-T1-AmpR(Positive control); N: Trans5α-pET28a-ACP-AmpR(Negative control); I: Trans5α-pET28a-I-2-AmpR. B: The OD values of Trans5α-pET28a-ACP-AmpR, Trans5α-pEASY-T1-AmpR and Trans5α-pET28a-I-2-AmpR in LB medium at 0, 25, and 40 μg/mL AMP, respectively
启动子片段 Promoter fragment | 启动子序列 Promoter sequence(5'-3') |
---|---|
I-2 | gcttgaatgcagcaagcaggcaactcgggcatcggagttgaccaatttggga caggcggaggtacatgtacccatgtagccatgcatacggccacctcccaag ataaatcacagctgatgatcagaccatattgctgagcatgttcgacaaacg ttttctgcgcagttagccgctatAAAAAtgttaatagaaggtaattcgatc gaaaccgtttgcaaataatattcagtgccacaagtcggtcttcaccggctc catacgattattcgcagatactgagacgacatactatctgttcattctcacctgaacatg |
Table 1 Sequence of litI promoter fragment I-2
启动子片段 Promoter fragment | 启动子序列 Promoter sequence(5'-3') |
---|---|
I-2 | gcttgaatgcagcaagcaggcaactcgggcatcggagttgaccaatttggga caggcggaggtacatgtacccatgtagccatgcatacggccacctcccaag ataaatcacagctgatgatcagaccatattgctgagcatgttcgacaaacg ttttctgcgcagttagccgctatAAAAAtgttaatagaaggtaattcgatc gaaaccgtttgcaaataatattcagtgccacaagtcggtcttcaccggctc catacgattattcgcagatactgagacgacatactatctgttcattctcacctgaacatg |
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