Difference between revisions of "Part:BBa K2933025"

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<partinfo>BBa_K2933025 parameters</partinfo>
 
<partinfo>BBa_K2933025 parameters</partinfo>
 
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===Usage and Biology==
+
===Usage and Biology===
 
It is a kind of β-lactamase, sharing 55% amino acid identity with NDM-1.It is first isolated from Erythrobacter litoralis HTCC2594.
 
It is a kind of β-lactamase, sharing 55% amino acid identity with NDM-1.It is first isolated from Erythrobacter litoralis HTCC2594.
 
==References==
 
  
  
 
===Molecular cloning===
 
===Molecular cloning===
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>
+
First, we used the vector pET28b-sumo to construct our expression plasmid. And then we converted the plasmid constructed to ''E. coli'' DH5α to expand the plasmid largely.
 
<p style="text-align: center;">
 
<p style="text-align: center;">
   [[File:TJUSLS China--Elbla2-1-PCR.png]]<br>
+
   [[File:TJUSLS China--Elbla2-1-PCR.png|600px]]<br>
'''Figure 1.'''  Left: The PCR result of Elbla2-1. Right: The verification results by enzyme digestion.<br>
+
'''Figure 1.'''  Left: The PCR result of ElblaII. Right: The verification results by enzyme digestion.<br>
 
</p>
 
</p>
 
After verification, it was determined that the construction is successful. We converted the plasmid to ''E. coli'' BL21(DE3) for expression and purification.<br>
 
After verification, it was determined that the construction is successful. We converted the plasmid to ''E. coli'' BL21(DE3) for expression and purification.<br>
Line 34: Line 32:
 
'''Pre-expression:'''<br>
 
'''Pre-expression:'''<br>
 
The bacteria were cultured in 5mL LB liquid medium with ampicillin(100 μg/mL final concentration) in 37℃ overnight.<br>
 
The bacteria were cultured in 5mL LB liquid medium with ampicillin(100 μg/mL final concentration) in 37℃ overnight.<br>
 
 
'''Massive expressing:'''<br>
 
'''Massive expressing:'''<br>
After taking samples, we transfer 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 0.3 mM IPTG (isopropylthiogalactoside, MW 238 g/mol) or ~0.1 gram per 1.5 liter flask. Put the liter flasks in 16°C shaking incubator for 16h. Centrifuge your bacteria in 500 mL bottles in the 4°C rotor at 4,000 RPM for 20 mins. Do this in batches until all your culture is spun down saving the cell pastes each time.<br>            
+
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>
 +
 
 +
'''Affinity Chromatography:'''<br>
 +
We used the Ni Agarose to purify the target protein. The Ni Agarose can combine specifically with the Ni-Sumo tag fused with target protein. <br>
 +
* First, wash the column with water for 10 minutes. Change to Ni-binding buffer for another 10 minutes and balance the Ni column.<br>
 +
* Second, add the protein solution to the column, let it flow naturally and bind to the column. <br>
 +
* Third, add Ni-Washing buffer several times and let it flow. Take 5ul of wash solution and test with Coomassie Brilliant Blue. Stop washing when it doesn’t turn blue.<br>
 +
* Forth, add Ni-Elution buffer several times. Check as above.<br>
 +
* Fifth, collect the eluted proteins for further operation. <br>
  
'''Purification of GST fusion proteins:'''<br>
 
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>
 
* First, wash the column with GST-binding buffer for 10 minutes to balance the GST column.<br>
 
* Second, add the protein solution to the column, let it flow naturally and bind to the column.<br>
 
* 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>
 
* Forth, add 400μL Prescission Protease (1mg/mL) to the agarose. Digest for 16 hours in 4℃.
 
* Fifth, add GST-Elution buffer several times. Check as above. Collect the eluted proteins for further operation.<br>
 
  
'''Anion exchange column:'''<br>
 
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 around 4.7 in theory, we choose buffer pH of 7.4 and use anion exchange column for purification.
 
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.
 
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>
 
  
 
'''Gel filtration chromatography:'''<br>
 
'''Gel filtration chromatography:'''<br>
Line 55: Line 49:
 
<p style="text-align: center;">
 
<p style="text-align: center;">
 
   [[File:TJUSLS China--Elbla2-1 gel.png]]<br>
 
   [[File:TJUSLS China--Elbla2-1 gel.png]]<br>
'''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 26kD.<br>
+
'''Figure 2.'''  (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 26kD.<br>
 
</p>
 
</p>
  
Line 74: Line 68:
 
====Determination of enzyme concentration====
 
====Determination of enzyme concentration====
 
<p style="text-align: center;">
 
<p style="text-align: center;">
     [[File:TJUSLS_China--Elbla2-1_enzyme_1.png]]<br>
+
     [[File:ELBLAtxgj.jpeg|300px|]][[File:TJUSLS_China--Elbla2-1_enzyme_1.png|300px|]]<br>
'''Figure 2.'''  The concentration of CDC-1 was fixed at 10.5 μM and the enzyme concentration was changed within a certain range, and the fluorescence value was measured with a function of reaction time. (a) First, we selected three gradient concentrations (with large intervals) for pre-experiment, and determined the gradient range of the formal experiment through the experimental results. (b) The appropriate enzyme concentration was selected for determination of the gradient, and the reaction curve of gradual rise was obtained.<br>
+
'''Figure 3.'''  The concentration of CDC-1 was fixed at 8.5 μM and the enzyme concentration was changed within a certain range, and the fluorescence value was measured with a function of reaction time. Left: First, we selected three gradient concentrations (with large intervals) for pre-experiment, and determined the gradient range of the formal experiment through the experimental results. Right: The appropriate enzyme concentration was selected for determination of the gradient, and the reaction curve of gradual rise was obtained.<br>
 
</p>
 
</p>
 
<p style="text-align: center;">
 
<p style="text-align: center;">
     [[File:TJUSLS China--Elbla2-1 enzyme 2.png]]<br>
+
     [[File:TJUSLS China--Elbla2-1 enzyme 2.png|300px|]]<br>
'''Figure 3.'''  We took the emission fluorescence at 3.02nm as the maximum emission fluorescence, and took the logarithm value of different NDM-23 enzyme concentrations to make the relationship curve between protein concentration and fluorescence emission rate. When the emittance of the system was 80%, the protein concentration was 1.51nM, that is, EC80 was 1.51nM.<br>
+
'''Figure 4.'''  We took the emission fluorescence at 30.2nm as the maximum emission fluorescence, and took the logarithm value of different NDM-23 enzyme concentrations to make the relationship curve between protein concentration and fluorescence emission rate. When the emittance of the system was 80%, the protein concentration was 2.273nM, that is, EC80 was 2.273nM.<br>
 
</p>
 
</p>
  
 
====Determination of the buffer condition====
 
====Determination of the buffer condition====
 
<p style="text-align: center;">
 
<p style="text-align: center;">
     [[File:TJUSLS China--Elbla2-1 buffer.png]]<br>
+
     [[File:TJUSLS China--Elbla2-1 buffer.png|600px|]]<br>
'''Figure 4.'''  Effect of different buffer condition on enzyme activity.<br>
+
'''Figure 5.'''  Effect of different buffer condition on enzyme activity.<br>
 
</p>
 
</p>
According to the experimental results, we chose NaCl concentration of 40mM, ZnCl concentration of 25 micron and pH of 8.5. <br>
+
According to the experimental results, we chose NaCl concentration of 35mM, ZnCl concentration of 25 micron and pH of 8.5. <br>
  
====Michaelis-Menten plot and Lineweaver-Burk plot====
+
====Michaelis-Menten plot====
 
<p style="text-align: center;">
 
<p style="text-align: center;">
     [[File:TJUSLS China--Elbla2-1 Km.png]]<br>
+
     [[File:Km of ElBlaII.jpeg|300px|]]<br>
'''Figure 5.'''  (a) The relationship between the substrate concentration and the maximum initial rate was obtained by using the Michaelis-Menten plot. (b) The relationship between the substrate concentration and the maximum initial rate was obtained by using the Lineweaver-Burk plot.<br>
+
'''Figure 6.'''  The relationship between the substrate concentration and the maximum initial rate was obtained by using the Michaelis-Menten plot.<br>
 
</p>
 
</p>
 
<p style="text-align: center;">
 
<p style="text-align: center;">
Line 100: Line 94:
 
<p style="text-align: center;">
 
<p style="text-align: center;">
  
     [[File:TJUSLS China--Elbla2-1 canshu.png]]<br>
+
     [[File:TJUSLS China--Elbla2-1 canshu.png|600px]]<br>
'''Figure 6.'''  The enzyme kinetic parameter of Elbla2-1.<br>
+
'''Figure 7.'''  The enzyme kinetic parameter of ElblaII.</p>
 +
 
 +
===Establishment of ElblaII inhibitor screening system===
 +
After the above determination of enzyme activity and the trial of concentration and buffer components, we determined the optimal conditions of Elbla2-1 enzyme activity and then established the screening system.<br>
 +
<p style="text-align: center;">
 +
[[File:TJUSLS China--Elbla2-1 tiaojian.png|600px]]<br>
 +
'''Figure8.'''  Protein concentration and optimal buffer components and the inhibitor screening system of ElblaII.
 
</p>
 
</p>
  
 
===Effective inhibitors in vitro we founded===
 
===Effective inhibitors in vitro we founded===
                      [[File:TJUSLS_China--Elbla2-1 inhibitor.png|600px]]
+
Above, we have established the NDM-23 high-throughput screening system, and then we used the microplate reader to conduct high-throughput screening to screen out nearly '''10''' inhibitors with significant inhibitory effect on NDM-23 from the drug library containing over '''4000''' small molecules.<br>
 +
 
 +
<p style="text-align: center;">
 +
[[File:TJUSLS_China--Elbla2-1 inhibitor.png|800px|]]
 +
[[File:E2.png|800px|]]
 +
[[File:E3.png|800px|]]
 +
[[File:ElBla4.png|800px|]]
 +
[[File:E5.png|800px|]]
 +
[[File:E6.png|800px|]]
 +
[[File:Zinc ElBla.png|800px|]]
 +
[[File:Bromfenac Sodium ElBla.png|800px|]]
 +
[[File:Isoconazole ElBla.png|800px|]]
 +
 
 +
===IC<sub>50</sub> and inhibitory mechanism of inhibitors===
 +
We tested the IC<sub>50</sub> of two inhibitors.<br>
 +
</p>
 +
<p style="text-align: center;">
 +
[[File:TA ElBla II IC50.jpeg|300px|]]<br>
 +
'''Figure 9.'''  IC<sub>50</sub> of Tannic acid for ElBlaII.<br>
 +
</p>
 +
<p style="text-align: center;">
 +
[[File:Isoconazole nitrate IC50.jpeg|300px|]]<br>
 +
'''Figure 10.''' IC<sub>50</sub> of Isoconazole nitrate for ElBlaII.<br>
 +
</p>
 +
 
 +
===Monitoring in living bacterial cells with antibiotics===
 +
After high-throughput screening, tannic acid was screened as the inhibitor of ElBlaII. We have used the UV visible method to assess the effectiveness of the treatment. The results are as follows:<br>
 +
<p style="text-align: center;">
 +
  [[File:Elbla TA.jpeg|300px]]<br>
 +
'''Figure 11.'''  Monitoring in living bacterial cells with antibiotics and Tannic acid.<br>
 +
</p>
 +
===Conclusion===
 +
In conclusion, beta-lactamase II [Erythrobacter litoralis HTCC2594] protein was successfully expressed in this part. We measured enzyme activity, established the high-throughput screening system, successfully screened some effective inhibitors with CDC-1 probes and then verified one of them with live bacteria to determine the IC<sub>50</sub> of the inhibitors in vivo. We found that inhibitors can effectively inhibit the activity of the enzyme in vivo and prevent the hydrolysis of cefazolin by the enzyme. We are proud that our results have laid the foundation for further research.
 +
 
 +
===References===
 +
[1] Girlich D, Poirel L, Nordmann P, Diversity of naturally occurring Ambler class B metallo-β-lactamases in Erythrobacter spp. The Journal of Antimicrobial Chemotherapy [31 Jul 2012, 67(11):2661-2664]

Latest revision as of 13:20, 21 October 2019


subclass B1 beta-lactamase II [Erythrobacter litoralis HTCC2594], codon optimized in E. coli

This part encodes a protein called beta-lactamase II [Erythrobacter litoralis HTCC2594], which is a metallo-beta-lactamase of subclass B1.

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
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 484
  • 1000
    COMPATIBLE WITH RFC[1000]


Usage and Biology

It is a kind of β-lactamase, sharing 55% amino acid identity with NDM-1.It is first isolated from Erythrobacter litoralis HTCC2594.


Molecular cloning

First, we used the vector pET28b-sumo to construct our expression plasmid. And then we converted the plasmid constructed to E. coli DH5α to expand the plasmid largely.

TJUSLS China--Elbla2-1-PCR.png
Figure 1. Left: The PCR result of ElblaII. Right: The verification results by enzyme digestion.

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

Expression and purification

Pre-expression:
The bacteria were cultured in 5mL LB liquid medium with ampicillin(100 μg/mL final concentration) in 37℃ overnight.
Massive expressing:
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.

Affinity Chromatography:
We used the Ni Agarose to purify the target protein. The Ni Agarose can combine specifically with the Ni-Sumo tag fused with target protein.

  • First, wash the column with water for 10 minutes. Change to Ni-binding buffer for another 10 minutes and balance the Ni column.
  • Second, add the protein solution to the column, let it flow naturally and bind to the column.
  • Third, add Ni-Washing buffer several times and let it flow. Take 5ul of wash solution and test with Coomassie Brilliant Blue. Stop washing when it doesn’t turn blue.
  • Forth, add Ni-Elution buffer several times. Check as above.
  • Fifth, collect the eluted proteins for further operation.


Gel filtration chromatography:
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.

TJUSLS China--Elbla2-1 gel.png
Figure 2. (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 26kD.

Enzyme activity determination

We used CDC-1, a probe with a similar structure from the beta lactam ring and a luminescent group for enzyme activity measurements. For more information on the substrate CDC-1, please see our project introduction.

Materials:
General 96-well plates (Black)
Infinite M1000 Pro Automatic Microplate Reader
Multi-channel adjustable pipette
Ultrasonic Cleaner

Buffer:
100% DMSO
Fluorescent Probe(CDC-1)
Target Enzyme(beta-lactamase)

Determination of enzyme concentration

ELBLAtxgj.jpegTJUSLS China--Elbla2-1 enzyme 1.png
Figure 3. The concentration of CDC-1 was fixed at 8.5 μM and the enzyme concentration was changed within a certain range, and the fluorescence value was measured with a function of reaction time. Left: First, we selected three gradient concentrations (with large intervals) for pre-experiment, and determined the gradient range of the formal experiment through the experimental results. Right: The appropriate enzyme concentration was selected for determination of the gradient, and the reaction curve of gradual rise was obtained.

TJUSLS China--Elbla2-1 enzyme 2.png
Figure 4. We took the emission fluorescence at 30.2nm as the maximum emission fluorescence, and took the logarithm value of different NDM-23 enzyme concentrations to make the relationship curve between protein concentration and fluorescence emission rate. When the emittance of the system was 80%, the protein concentration was 2.273nM, that is, EC80 was 2.273nM.

Determination of the buffer condition

TJUSLS China--Elbla2-1 buffer.png
Figure 5. Effect of different buffer condition on enzyme activity.

According to the experimental results, we chose NaCl concentration of 35mM, ZnCl concentration of 25 micron and pH of 8.5.

Michaelis-Menten plot

Km of ElBlaII.jpeg
Figure 6. The relationship between the substrate concentration and the maximum initial rate was obtained by using the Michaelis-Menten plot.

Calculate Km, Vm with the Lineweaver-Burk plot, because it fit better. Kcat values were calculated with the results of maximum fluorescence values at different substrate concentrations.

TJUSLS China--Elbla2-1 canshu.png
Figure 7. The enzyme kinetic parameter of ElblaII.

Establishment of ElblaII inhibitor screening system

After the above determination of enzyme activity and the trial of concentration and buffer components, we determined the optimal conditions of Elbla2-1 enzyme activity and then established the screening system.

TJUSLS China--Elbla2-1 tiaojian.png
Figure8. Protein concentration and optimal buffer components and the inhibitor screening system of ElblaII.

Effective inhibitors in vitro we founded

Above, we have established the NDM-23 high-throughput screening system, and then we used the microplate reader to conduct high-throughput screening to screen out nearly 10 inhibitors with significant inhibitory effect on NDM-23 from the drug library containing over 4000 small molecules.

TJUSLS China--Elbla2-1 inhibitor.png E2.png E3.png ElBla4.png E5.png E6.png Zinc ElBla.png Bromfenac Sodium ElBla.png Isoconazole ElBla.png

IC50 and inhibitory mechanism of inhibitors

We tested the IC50 of two inhibitors.

TA ElBla II IC50.jpeg
Figure 9. IC50 of Tannic acid for ElBlaII.

Isoconazole nitrate IC50.jpeg
Figure 10. IC50 of Isoconazole nitrate for ElBlaII.

Monitoring in living bacterial cells with antibiotics

After high-throughput screening, tannic acid was screened as the inhibitor of ElBlaII. We have used the UV visible method to assess the effectiveness of the treatment. The results are as follows:

Elbla TA.jpeg
Figure 11. Monitoring in living bacterial cells with antibiotics and Tannic acid.

Conclusion

In conclusion, beta-lactamase II [Erythrobacter litoralis HTCC2594] protein was successfully expressed in this part. We measured enzyme activity, established the high-throughput screening system, successfully screened some effective inhibitors with CDC-1 probes and then verified one of them with live bacteria to determine the IC50 of the inhibitors in vivo. We found that inhibitors can effectively inhibit the activity of the enzyme in vivo and prevent the hydrolysis of cefazolin by the enzyme. We are proud that our results have laid the foundation for further research.

References

[1] Girlich D, Poirel L, Nordmann P, Diversity of naturally occurring Ambler class B metallo-β-lactamases in Erythrobacter spp. The Journal of Antimicrobial Chemotherapy [31 Jul 2012, 67(11):2661-2664]