Difference between revisions of "Part:BBa K2933244"

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===Usage and Biology===
 
===Usage and Biology===
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This composite part is made up with three basic parts, the GST tag, the cutting site of Prescission Protease and our target protein NDM-23. It encodes a protein which is NDM-23 fused with GST tag. The fusion protein is about 40.5 kD. In order to gain the highly purified target protein, we add GST tag in N-terminal of NDM-23 and combine the two parts with the cutting site of Prescission Protease. The fusion protein can be cut off at the cutting site by Prescission Protease. It is convenient for us to purify our target protein.<br>
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This composite part is made up with eight basic parts, T7 Ribosome binding sites,the His-Sumo tag, three cutting sites of Prescission Protease, our target protein NDM-23 and T7 terminator. It encodes a protein which is NDM-23 fused with His-Sumo tag. The fusion protein is about 40.5 kD. The fusion protein can be cut off at the cutting sites by Prescission Protease. It is convenient for us to purify our target protein.<br>
  
 
===Molecular cloning===
 
===Molecular cloning===
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First, we used the vector pGEX-6p-1 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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First, we used the vector pET-28bs to construct our expression plasmid. And then we converted the plasmid constructed to ''E. coli'' DH5α to expand the plasmid largely.<br>
 
<p style="text-align: center;">
 
<p style="text-align: center;">
 
   [[File:NDM-23-PCR.png|500px]]<br>
 
   [[File:NDM-23-PCR.png|500px]]<br>
 
'''Figure 1.'''  Left: The PCR result of NDM-23. Right: The verification results by enzyme digestion.<br>
 
'''Figure 1.'''  Left: The PCR result of NDM-23. Right: The verification results by enzyme digestion.<br>
 
</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>
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===References===
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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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===Expression and purification===
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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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'''Pre-expression:'''<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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The bacteria were cultured in 5mL LB liquid medium with ampicillin(100 μg/mL final concentration) in 37℃ overnight.<br>
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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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'''Massive expressing:'''<br>
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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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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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[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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[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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'''Affinity Chromatography:'''<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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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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[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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* First, wash the column with GST-binding buffer for 10 minutes to balance the GST column.<br>
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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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* Second, add the protein solution to the column, let it flow naturally and bind to the column.<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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* 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;">
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    [[File:T--TJUSLS China--NDM 23 GST.jpg|400px]]<br>
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'''Figure 2.'''  The result of SDS-page.<br>
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</p>
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'''Anion exchange column:'''<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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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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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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<p style="text-align: center;">
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  [[File:T--TJUSLS China--NDM 23 Q.jpg|400px]]<br>
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'''Figure 1.'''  The result of SDS-page of superdex75 Q column.<br>
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</p>
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'''Gel filtration chromatography:'''<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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</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>
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Latest revision as of 14:43, 25 September 2019


RBS b+Linker h+His+Linker a+Sumo+Linker b+NDM-23+T7 terminator

This part consists of RBS, protein coding sequence(His+Linker a+Sumo+Linker b+NDM-23) and T7 terminator,and the biological module can be build into E.coli for protein expression. This part can be prefaced with promoters of different strengths and types to regulate expression function.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 298
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 298
    Illegal NheI site found at 75
    Illegal NheI site found at 1218
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 298
    Illegal BglII site found at 187
    Illegal BamHI site found at 386
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 298
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 298
  • 1000
    COMPATIBLE WITH RFC[1000]


Usage and Biology

This composite part is made up with eight basic parts, T7 Ribosome binding sites,the His-Sumo tag, three cutting sites of Prescission Protease, our target protein NDM-23 and T7 terminator. It encodes a protein which is NDM-23 fused with His-Sumo tag. The fusion protein is about 40.5 kD. The fusion protein can be cut off at the cutting sites by Prescission Protease. It is convenient for us to purify our target protein.

Molecular cloning

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

NDM-23-PCR.png
Figure 1. Left: The PCR result of NDM-23. Right: The verification results by enzyme digestion.

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.