Difference between revisions of "Part:BBa K2933203"
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<partinfo>BBa_K2933203 parameters</partinfo>
 
<partinfo>BBa_K2933203 parameters</partinfo>
 
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==Usage and Biology===
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==Usage and Biology==
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This composite part is made up with three basic parts, the His tag, T7 promoter, RBS and our target protein NDM-23. It encodes a protein which is NDM-23 fused with His tag. The fusion protein is about 28.5 kD. In order to gain the highly purified target protein, we add His tag in N-terminal of NDM-23 .  It is convenient for us to purify our target protein.<br>
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This composite part is made up with seven basic parts, the His tag, T7 promoter, RBS ，Linker h ，Linker f，T7 terminator and our target protein NDM-23. It encodes a protein which is NDM-23 fused with His tag. Linker h is from the vector pET-28a, which connects the RBS to His tag sequence.Linker f from vector pGEX-6p-1, contains the thrombin restriction site and T7 tag.The fusion protein is about 28.5 kD. In order to gain the highly purified target protein, we add His tag in N-terminal of NDM-23 .  It is convenient for us to purify our target protein.<br>
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===Expression and purification===
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===Molecular cloning===
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'''Pre-expression:'''<br>
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First, we used the vector pGEX-28a to construct our expression plasmid. And then we converted the plasmid constructed to ''E. coli'' DH5α to expand the plasmid largely.<br>
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The bacteria were cultured in 5mL LB liquid medium with ampicillin(100 μg/mL final concentration) in 37℃ overnight.<br>
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'''Massive expressing:'''<br>
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After taking samples, we transfered them into 1L LB medium and add antibiotic to 100 μg/mL final concentration. Grow them up in 37°C shaking incubator. Grow until an OD 600 nm of 0.8 to 1.2 (roughly 3-4 hours). Induce the culture to express protein by adding 1 mM IPTG (isopropylthiogalactoside, MW 238 g/mol). Put the liter flasks in 16°C shaking incubator for 16h.<br>
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'''Affinity Chromatography:'''<br>
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We used the GST Agarose to purify the target protein. The GST Agarose can combine specifically with the GST tag fused with target protein. <br>
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* First, wash the column with GST-binding buffer for 10 minutes to balance the GST column.<br>
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* Second, add the protein solution to the column, let it flow naturally and bind to the column.<br>
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* Third, add GST-Washing buffer several times and let it flow. Take 10μl of wash solution and test with Coomassie Brilliant Blue. Stop washing when it doesn’t turn blue.<br>
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* Forth, add 400μL Prescission Protease (1mg/mL) to the agarose. Digest for 16 hours in 4℃.
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* Fifth, add GST-Elution buffer several times. Check as above. Collect the eluted proteins for further operation.<br>
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<p style="text-align: center;">
 
<p style="text-align: center;">
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    [[File:T--TJUSLS China--NDM 23 GST1.jpg|400px]]<br>
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  [[File:NDM-23-PCR1.png|500px]]<br>
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===References===
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'''Figure 2.'''  The result of SDS-page.<br>
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[1] Van Duin D, Doi Y. The global epidemiology of carbapenemase-producing Enterobacteriaceae [J]. Virulence, 2017,8(4): 460469.<br>
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</p>
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[2] Yong D, Toleman MA, Giske cG, et al. Characterization of a new metallo-beta-lactamase gene, bla(NDM-1), and a novel erythromycin esterase gene carried on a unique geneticstructure in Klebsiella pneumoniae sequence type 14 from India [J]. Antimicrob Agents Chemother, 2009,53(12): 5046-5054.<br>
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'''Anion exchange column:'''<br>
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[3] Wu w. Feng Y, Tang G et al. NDM Metallo-ß-Lactamases and Their Bacterial Producers in Health Care Sttings [J]. Clin Microbiol Rev, 2019,32(2): 0011500118.<br>
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According to the predicted pI of the protein and the pH of the ion-exchange column buffer, firstly select the appropriate ion exchange column (anion exchange column or cation exchange column). The pH of buffer should deviate from the isoelectric point of the protein. Since the isoelectric point of our protein is 5.88 in theory, we choose buffer pH of 7.4 and use anion exchange column for purification.  
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[4] Khan AU, Maryam L, Zarilli R. Structure, Genetics and Worldwide Spread of New Delhi Maeallo-beta-lactamase (NDM): a threat to public health [J].BMC Microbiol, 2017,17(1):101-112.<br>
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The protein is concentrated with a 10KD concentration tube, and then the exchange buffer is used to exchange the protein to the ion-exchange liquid A. Finally, it is concentrated to less than 5ml by centrifuging at 4℃ and 3400rpm for 10 minutes in a high-speed centrifuge to remove insoluble substances and bubbles.
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[5] Zheng B, Lv T, Xu H, et al. Discovery and characterisation of an escherichia coli ST206 strain producing NDM-5 and MCR-1 from a patient with acute diarrhoea in China [J]. Int JAntimicrob Agents, 2018,51(2): 273-275.<br>
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Balance the selected column with liquid A. Through the AKTApure protein purification system, the samples are loaded to the column at a flow rate of 0.5ml/min, and continue washing for 5min. Gradually increase the content of liquid B in the column, change the salt concentration and then change the interaction between the sample and the column, and collect the corresponding eluent according to the position of the peak. Use SDS-PAGE to check the result.<br>
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[6] Li X, Jiang Y, Wu K,et al. Whole-genome sequencing identification of a multidrug-resistan t Salmonella enterica serovar Typhimurium strain carrying blaNDM-5 from Guangdong, China [J]. Infect Genet Evol, 2017,55: 195-198.<br>
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<p style="text-align: center;">
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[7] Rahman M, Shukla SK, Prasad KN, et al. Prevalence and molecular characterisation of New Delhi metallo-β-lactamases NDM-I, NDM-5, NDM-6 and NDM-7 in multidrug- resistant Enterobacteriaceae from India [J]. Int J Antimierob Agents, 2014，44(1).<br>
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  [[File:T--TJUSLS China--NDM 23 Q.jpg|400px]]<br>
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[8] Rojas LJ, Hujer AM, Rudin SD, et al. NDM-5 and OXA-181 Beta-Lactamases, a Significant Threat Continues To Spread in the Americas [J]. Antimicrob Agents Chemother,2017,61(7): pii: e00454-17. <br>
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'''Figure 1.'''  The result of SDS-page of superdex75 Q column.<br>
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[9] Almakki A, Maure A, Pantel A, et al. NDM-5-producing Escherichia coli in an urban river in Montpellier, France [I]. Int J Antimicrob Agents, 2017,50(1): 123-124.<br>
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</p>
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[10] Rozales FP, Magagnin cM, Campos JC, et al. Characterization of Transformants Obtained From NDM-1-Producing Enterobacteriaceae in Brazil [J]. Infect Control Hosp Epidemiol,2017,38(5): 634-636.<br>
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'''Gel filtration chromatography:'''<br>
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[11] Yang B, Feng Y, McNally A, et al. Occurrence of Enterobacter hormaechei carrying blaNDM-1 and blaKPC-2 in China [J]. Diagn Microbiol Infect Dis, 2018.90(2): 139-142.<br>
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The collected protein samples are concentrated in a 10 KD concentrating tube at a speed of 3400 rpm and concentrated for a certain time until the sample volume is 500 μl. At the same time, the superdex 200 column is equilibrated with a buffer to balance 1.2 column volumes. The sample is then loaded and 1.5 cylinders are eluted isocratically with buffer. Determine the state of protein aggregation based on the peak position and collect protein samples based on the results of running the gel.<br>
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'''Figure 1.'''  The PCR result of NDM-23. <br>
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</p>
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<p style="text-align: center;">
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  [[File:T--TJUSLS China--NDM 23 gel jiaotu+fengtu.png|600px]]<br>
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'''Figure 1.'''  (a) The result of gel filtration used the superdex75 column with the AKTA system, which shows that the target protein is monomeric. (b) The result of SDS-PAGE. And the target protein is about 28.5kD.<br>
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</p>
 
</p>
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After verification, it was determined that the construction is successful. We converted the plasmid to ''E. coli'' BL21(DE3) for expression and purification.<br>

Latest revision as of 14:00, 23 September 2019


T7 promoter+RBS b+Linker h+His+Linker f+NDM-23+T7 terminator

This part consists of T7 promoter, RBS and protein coding sequence（His+Linker f+NDM-23），and the biological module can be built into E.coli for protein expression.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal XbaI site found at 47
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 169
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal XbaI site found at 47
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal XbaI site found at 47
  • 1000
    COMPATIBLE WITH RFC[1000]


Usage and Biology

This composite part is made up with seven basic parts, the His tag, T7 promoter, RBS ，Linker h ，Linker f，T7 terminator and our target protein NDM-23. It encodes a protein which is NDM-23 fused with His tag. Linker h is from the vector pET-28a, which connects the RBS to His tag sequence.Linker f from vector pGEX-6p-1, contains the thrombin restriction site and T7 tag.The fusion protein is about 28.5 kD. In order to gain the highly purified target protein, we add His tag in N-terminal of NDM-23 . It is convenient for us to purify our target protein.

Molecular cloning

First, we used the vector pGEX-28a to construct our expression plasmid. And then we converted the plasmid constructed to E. coli DH5α to expand the plasmid largely.

NDM-23-PCR1.png

References

[1] Van Duin D, Doi Y. The global epidemiology of carbapenemase-producing Enterobacteriaceae [J]. Virulence, 2017,8(4): 460469.
[2] Yong D, Toleman MA, Giske cG, et al. Characterization of a new metallo-beta-lactamase gene, bla(NDM-1), and a novel erythromycin esterase gene carried on a unique geneticstructure in Klebsiella pneumoniae sequence type 14 from India [J]. Antimicrob Agents Chemother, 2009,53(12): 5046-5054.
[3] Wu w. Feng Y, Tang G et al. NDM Metallo-ß-Lactamases and Their Bacterial Producers in Health Care Sttings [J]. Clin Microbiol Rev, 2019,32(2): 0011500118.
[4] Khan AU, Maryam L, Zarilli R. Structure, Genetics and Worldwide Spread of New Delhi Maeallo-beta-lactamase (NDM): a threat to public health [J].BMC Microbiol, 2017,17(1):101-112.
[5] Zheng B, Lv T, Xu H, et al. Discovery and characterisation of an escherichia coli ST206 strain producing NDM-5 and MCR-1 from a patient with acute diarrhoea in China [J]. Int JAntimicrob Agents, 2018,51(2): 273-275.
[6] Li X, Jiang Y, Wu K,et al. Whole-genome sequencing identification of a multidrug-resistan t Salmonella enterica serovar Typhimurium strain carrying blaNDM-5 from Guangdong, China [J]. Infect Genet Evol, 2017,55: 195-198.
[7] Rahman M, Shukla SK, Prasad KN, et al. Prevalence and molecular characterisation of New Delhi metallo-β-lactamases NDM-I, NDM-5, NDM-6 and NDM-7 in multidrug- resistant Enterobacteriaceae from India [J]. Int J Antimierob Agents, 2014，44(1).
[8] Rojas LJ, Hujer AM, Rudin SD, et al. NDM-5 and OXA-181 Beta-Lactamases, a Significant Threat Continues To Spread in the Americas [J]. Antimicrob Agents Chemother,2017,61(7): pii: e00454-17.
[9] Almakki A, Maure A, Pantel A, et al. NDM-5-producing Escherichia coli in an urban river in Montpellier, France [I]. Int J Antimicrob Agents, 2017,50(1): 123-124.
[10] Rozales FP, Magagnin cM, Campos JC, et al. Characterization of Transformants Obtained From NDM-1-Producing Enterobacteriaceae in Brazil [J]. Infect Control Hosp Epidemiol,2017,38(5): 634-636.
[11] Yang B, Feng Y, McNally A, et al. Occurrence of Enterobacter hormaechei carrying blaNDM-1 and blaKPC-2 in China [J]. Diagn Microbiol Infect Dis, 2018.90(2): 139-142.
Figure 1. The PCR result of NDM-23.

After verification, it was determined that the construction is successful. We converted the plasmid to E. coli BL21(DE3) for expression and purification.