Difference between revisions of "Part:BBa K3078005"
Line 17: Line 17:
  
 
<h1>'''2. Characterization'''</h1>
 
<h1>'''2. Characterization'''</h1>
−
<h4>'''2.1 Validation of pVE-&#946;-1,3-glucanase construction'''</h4>
+
<h4>'''2.1 Validation of pVE-LL-37 construction'''</h4>
 
<h5>
 
<h5>
 
<P style="text-indent:2em;">
 
<P style="text-indent:2em;">
−
We characterised &#946;-1,3-glucanase by cloning it into pVE vector. Moreover, an signal peptide were added.
+
To verify the construction of pVE-LL-37 which we generated, the digestion by BglII/EcoRV was performed by a standard protocol following agarose gel electrophoresis (Figure 1).
−
</p>
+
−
<P style="text-indent:2em;">
+
−
To verify the construction of pVE-&#946;-1,3-glucanase(pVE-&#946;-GA) which we generated, the digestion by SalI/EcoRV was performed by a standard protocol following agarose gel electrophoresis (Figure 1).
+
 
</p>
 
</p>
 
</h5>
 
</h5>
−
[[File:B13-1.png|center|B13-1]]
+
[[File:L13-1.png|center|L13-1]]
 
<center>
 
<center>
−
Figure 1. Digestion and agarose gel electrophoresis of pVE-&#946;-GA.
+
Figure 1. Digestion and agarose gel electrophoresis of pVE-LL-37.
 
</center>
 
</center>
  
Line 34: Line 31:
  
  
−
<h4>'''2.2 Expression of pVE-&#946;-1,3-glucanase '''</h4>
+
<h4>'''2.2 Expression of pVE-LL-37 '''</h4>
 
<h5>
 
<h5>
 
<P style="text-indent:2em;">
 
<P style="text-indent:2em;">
−
To assess the expression of our &#946;-1,3-glucanase, Congo Red experiment was used.
+
To detect the expression of pVE-LL-37 in E. coli, the constructs were transformed into BL21. Compared to the mock, there was a small size band expressed in pVE-LL-37 (Figure 2).
 
</p>
 
</p>
 +
</h5>
 +
[[File:L13-2.png|center|L13-2]]
 +
<center>
 +
Figure 2. Expression of pVE-LL-37. The bacteria were collected and ultrasonication. The lysate was centrifuged and supernate was electrophoresed on the Tricine-SDS-PAGE gel, followed by Fast silver staining. The LL-37 peptide was synthesized as positive control.
 +
</center>
 +
 +
 +
<h4>'''2.3 Candidacidal Effect of LL-37 '''</h4>
 +
<h5>
 
<P style="text-indent:2em;">
 
<P style="text-indent:2em;">
−
Congo Red has a strong chromogenic reaction with &#946;-1, 3-glucan, and when &#946;-1, 3-glucan is decomposed into reducing monosaccharides by &#946;-1, 3-glucanase, the hydrolyzed region forms a pale yellow transparent hydrolytic circle. The results of activity detection of Congo Red hydrolytic ring (Figure 2) showed that compared to the control, there was a larger size of transparent hydrolytic circle caused by &#946;-1, 3-glucanase expressed in E. coli with pVE-&#946;-GA.
+
To assess the candidacidal activity of LL-37, the spot assay was performed. As shown in Figure 3, C. albicans viability after LL-37 treatment was visually compared with that of control cells (no LL-37 treatment). We found that cell viability was more sensitive to LL-37.
 
</p>
 
</p>
 
</h5>
 
</h5>
−
[[File:B13-2.png|center|B13-2]]
+
[[File:L13-3.png|center|L13-3]]
 
<center>
 
<center>
−
Figure 2. Expression of pVE-&#946;-GA. Add 10 mg/ml Congo Red solution to LB medium containing &#946;-1,3-glucan substrate (0.1 g/100 mL) at a ratio of 1:100. 100 μl supernatant obtained by centrifugation after ultrasonic crushing of E. coli with pVE-&#946;-GA is added to the Oxford cup, and the pVE empty vector bacteria supernatant is used as the control. Stand at 37 ℃ for 24 hours.
+
Figure 3. Candidacidal activity of LL-37. C. albicans were collected and suspensions were resuspended to 105 cells/ml. 10 µl C. albicans suspension and 20 µl sterile BL21-transformed bacteria were gently mixed. After a 10-min incubation, the mixture was spotted onto the SDA agar plates. Cell viability was detected after incubation at 30℃ for 18 hrs.
 
</center>
 
</center>
  
−
 
−
<h4>'''2.3 Effect of &#946;-1,3-glucanase on biofilm in 96-well microplate  '''</h4>
 
 
<h5>
 
<h5>
 
<P style="text-indent:2em;">
 
<P style="text-indent:2em;">
−
Crystal violet(CV) reduction method, which is commonly used for quantitative analysis of biofilm, was used to evaluate the antibiofilm activity of &#946;-1,3-glucanase. The result indicates that &#946;-1,3-glucanase has disruption effect on mature biofilm with concentration-dependent manner. Under the condition of using bacteria with pVE empty vector to exclude the influence of bacterial substances on the staining results, as Figure 3 shows, &#946;-1,3-glucanase produced by our engineered bacteria has the effect of degrading biofilm. The supernatant of E. coli with pVE-&#946;-GA diluted by one time is estimated to reach the effect of 0.5 μg/mL~2 μg/mL samples of standard &#946;-1,3-glucanase.
+
To further validate the Candidacidal effect of LL-37, C. albicans suspensions were incubated with it. After incubation, LL-37 caused an immediate increase in PI fluorescence indicates that LL-37 can kill C. albicans.
 
</p>
 
</p>
 
</h5>
 
</h5>
−
[[File:B13-3.png|center|B13-3]]
+
[[File:L13-4.png|center|L13-4]]
 
<center>
 
<center>
−
Figure 3. Effect of &#946;-1,3-glucanase on biofilm in 96-well microplate. Biofilm formed in RPMI 1640 medium for 48 h. Mature biofilm was treated with RPMI 1640 medium, supernatant of bacteria with pVE empty vector or pVE-&#946;-GA and standard &#946;-1,3-glucanase in different concentrations (0.5, 1 and 2 μg/mL) for another 24 h. Values obtained are given as the percentage of biofilm. The experiment was performed three times in triplicate. *, P < 0.05 from mock control using Student’s t test. △, P < 0.05.
+
Figure 4. Candidacidal activity of LL-37. 1.5 µM PI (propidiumiodide) was added into C. albicans suspensions (106 cells/ml, 1 ml) and serially BL21-transformed bacteria supernatant (2 ml) mixture. The whole system was incubated at 30℃. During the 1 h incubation, the fluorescence was measured every 5 min at the excitation wavelength λexc 544 nm and emission wavelength λem 620 nm. The experiment was performed three times in triplicate. *, P < 0.05 from control using Student’s t test.
 
</center>
 
</center>
  

Revision as of 03:32, 21 October 2019

LL-37

LL37 protein coding region. We added a stop codon to BBa_K1162006.

1. Usage and Biology

LL-37 is an antimicrobial peptide (AMP) from the cathelicidin family of antimicrobial peptides, isolated from the Human (Homo sapiens). It is a 37-residue helical peptide found throughout the human body, exhibiting a broad spectrum of antimicrobial activity as well as a variety of immunomodulating effects (Dürr, Ulrich H.N., et. al 2006). It has the distinction of being the first and only cathelicidin member discovered from humans, and is extremely well studied and characterized for this reason. The peptide is produced via epithelial cells as well as leukocytes in places such as the skin, gastrointestinal tract, and the respiratory tract. This AMP is is cleaved from a larger protein, hCAP-18, in a series of modifications to yield its active form (Sorensen, O.E, et. al 2001).


2. Characterization

2.1 Validation of pVE-LL-37 construction

To verify the construction of pVE-LL-37 which we generated, the digestion by BglII/EcoRV was performed by a standard protocol following agarose gel electrophoresis (Figure 1).

L13-1

Figure 1. Digestion and agarose gel electrophoresis of pVE-LL-37.



2.2 Expression of pVE-LL-37

To detect the expression of pVE-LL-37 in E. coli, the constructs were transformed into BL21. Compared to the mock, there was a small size band expressed in pVE-LL-37 (Figure 2).

L13-2

Figure 2. Expression of pVE-LL-37. The bacteria were collected and ultrasonication. The lysate was centrifuged and supernate was electrophoresed on the Tricine-SDS-PAGE gel, followed by Fast silver staining. The LL-37 peptide was synthesized as positive control.


2.3 Candidacidal Effect of LL-37

To assess the candidacidal activity of LL-37, the spot assay was performed. As shown in Figure 3, C. albicans viability after LL-37 treatment was visually compared with that of control cells (no LL-37 treatment). We found that cell viability was more sensitive to LL-37.

L13-3

Figure 3. Candidacidal activity of LL-37. C. albicans were collected and suspensions were resuspended to 105 cells/ml. 10 µl C. albicans suspension and 20 µl sterile BL21-transformed bacteria were gently mixed. After a 10-min incubation, the mixture was spotted onto the SDA agar plates. Cell viability was detected after incubation at 30℃ for 18 hrs.

To further validate the Candidacidal effect of LL-37, C. albicans suspensions were incubated with it. After incubation, LL-37 caused an immediate increase in PI fluorescence indicates that LL-37 can kill C. albicans.

L13-4

Figure 4. Candidacidal activity of LL-37. 1.5 µM PI (propidiumiodide) was added into C. albicans suspensions (106 cells/ml, 1 ml) and serially BL21-transformed bacteria supernatant (2 ml) mixture. The whole system was incubated at 30℃. During the 1 h incubation, the fluorescence was measured every 5 min at the excitation wavelength λexc 544 nm and emission wavelength λem 620 nm. The experiment was performed three times in triplicate. *, P < 0.05 from control using Student’s t test.



3. Conclusion

Our engineered bacteria successfully characterized β-1,3-glucanase,moreover, it was effective in performing the function of degrading C. albicans biofilm.










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 NgoMIV site found at 274
    Illegal NgoMIV site found at 483
    Illegal NgoMIV site found at 622
  • 1000
    COMPATIBLE WITH RFC[1000]