Difference between revisions of "Part:BBa K3378888"

Revision as of 17:01, 27 October 2020

LRAP(Leucine-rich Amelogenin Peptides)

Leucine-rich ameloblast peptide (LRAP) is an alternative splicing product expressed by ameloblast genes during enamel development. LRAP has N-terminal amino acids and C-terminal amino acids of whole amelogenin, so it has the similar characteristics to the total length of amelogenin.

Usage and Biology

LRAP can self-assemble into a chain structure composed of nanospheres by relying on its C-terminal domain, and the presence of calcium ions can enhance its ability. Non-phosphorylated LRAP tends to induce the formation of ordered hydroxyapatite (HA), while phosphorylated LRAP tend to stabilize amorphous calcium phosphate (ACP) and prevent their conversion to hydroxyapatite in vitro. So the non-phosphorylation LRAP are sufficient to transform ACP into ordered bundles of apatite crystals.We also found that LRAP has the ability to bind to natural teeth. 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 132
1000
COMPATIBLE WITH RFC[1000]

Functional Parameters

To obtain the LRAP protein, we constructed pET-28a(+)-LRAP and transferred it to E. coli BL21(DE3) (Figure 1). The E. coli strain was cultured to OD₆₀₀~0.6, induced with 0.5 mM IPTG, and allowed to grow overnight at 16 ℃. LRAP’s molecular weight is about 6.5kDA. We infered that LRAP can be expressed in E. coli at the condition described above by Tris-Tricine-SDS-PAGE analysis(Figure 2).

Figure 1. Linear pET-28a(+) and LRAP obtained by PCR .

Figure 2. Tris-Tricine-SDS-PAGE analysis of ClyR expression
Since LRAP protein was difficult to be purified, we cultured BL21(DE3) strain without plasmid (WT), uninduced BL21(DE3) strain with plasmid(-IPTG) and induced BL21(DE3) strain with plasmid(+IPTG).And then crushed the bacteria by ultrasonication, centrifuged at 10000xg and 4℃ for 10min, and took the supernatant for follow-up experiments. To demonstrate the mineralization capacity of LRAP, the supernatant was added into artificial saliva(8.7mM KCl,0.6mM MgCl₂,10.mM CaCl₂,4.6mM K₂HPO₄,2.7mM KH₂PO₄) to make the final protein concentration was 5mg/ml. The solution was incubated at 37℃ for 24h and the turbidity was observed(Figure 3)We observed that the +IPTG group had less precipitation and higher turbidity and we speculated the phenomenon is because that the formation of precipitation is prevented by the nucleation of LRAP and ions in artificial saliva.

Figure 3.Effects of different supernatants on turbidity of artificial Saliva.
We incubated the supernatant of ultrasound-broken bacteria which include LRAP together with pig teeth. Then we designed the experiment called “Toothtern-blot” to verify the existence and function of LRAP. The teeth of domestic pigs were broken into fragments about 5mm long on each side, which were selected and stored in 75% alcohol at room temperature. The total protein concentration of the three supernatants was adjusted into 10mg/mL. Three pieces of pig teeth were washed with PBS buffer to remove the surface’s alcohol, then put in three 2mL centrifuge tubes respectively. After that, three supernatants with uniform protein concentration were added respectively. Incubate at 37℃ for 20h, and the tubes were upside down for several times every 5h to eliminate the influence of protein deposition processes as much as possible. The incubated pig teeth fragments were repeatedly washed with PBS buffer for 2-3min to remove the protein that was not bound to the teeth.The rinsed pig teeth were incubated in plastic petri dishes with diameter of 10cm, diluted with 50 mL 5% skimmed milk at 1:10000 and hybridized with His-Tag, Rabbit pAb (YEASEN Biotech,Shanghai, China) at room temperature for 2h. Then washed them with TBST buffer 3 times for 10min each. After that, diluted with 50 mL 5% skimmed milk at 1:10000 and hybridized with peroxidase-conjugated Goat anti-rabbit IgG (H+L) (YEASEN Biotech,Shanghai, China) at room temperature for 2h. Then washed them with TBST buffer 3 times for 10min each. Use A Super ECL Detection Reagent ECL (YEASEN Biotech,Shanghai, China)for development operations. The results showed that LRAP was able to bind to the tooth fragments and had binded more in the dentin(Figure 4).

Figure 4.”Toothtern-blot” analysis of different supernatants. A and B . The same result in different exposures. C. Picture of the teeth in reality.

@@ Line 5: / Line 5: @@
 Leucine-rich ameloblast peptide (LRAP) is an alternative splicing product expressed by ameloblast genes during enamel development. LRAP has N-terminal amino acids and C-terminal amino acids of whole amelogenin, so it has the similar characteristics to the total length of amelogenin.
-<!-- Add more about the biology of this part here
 ===Usage and Biology===
 LRAP can self-assemble into a chain structure composed of nanospheres by relying on its C-terminal domain, and the presence of calcium ions can enhance its ability. Non-phosphorylated LRAP tends to induce the formation of ordered hydroxyapatite (HA), while phosphorylated LRAP  tend to stabilize amorphous calcium phosphate (ACP) and prevent their conversion to hydroxyapatite in vitro. So the non-phosphorylation LRAP are sufficient to transform ACP into ordered bundles of apatite crystals.We also found that LRAP has the ability to bind to natural teeth.
@@ Line 15: / Line 14: @@
 <partinfo>BBa_K3378888 parameters</partinfo>
 <!-- -->
+To obtain the LRAP protein, we constructed pET-28a(+)-LRAP and transferred it to E. coli BL21(DE3) (<b>Figure 1</b>). The <i>E. coli</i> strain was cultured to OD<sub>600</sub>~0.6, induced with 0.5 mM IPTG, and allowed to grow overnight at 16 ℃. LRAP’s molecular weight is about 6.5kDA. We infered that LRAP can be expressed in <i>E. coli</i> at the condition described above by Tris-Tricine-SDS-PAGE analysis(<b>Figure 2</b>).
+<html>
+<body>
+<div style="width: 40%; margin: 30px auto">
+<img src=https://2020.igem.org/wiki/images/3/37/T--HZAU-China--LRAPF1.jpg width=100% alt="">
+</div>
+<br>
+<b>Figure 1.</b> Linear pET-28a(+) and LRAP obtained by PCR .
+<br>
+<img src=https://2020.igem.org/wiki/images/c/c7/T--HZAU-China--LRAPF2.jpg width=100% alt="">
+<br>
+<b>Figure 2.</b> Tris-Tricine-SDS-PAGE analysis of ClyR expression
+<br>
+Since LRAP protein was difficult to be purified, we cultured BL21(DE3) strain without plasmid (WT), uninduced BL21(DE3) strain with plasmid(-IPTG) and induced BL21(DE3) strain with plasmid(+IPTG).And then crushed the bacteria by ultrasonication, centrifuged at 10000xg and 4℃ for 10min, and took the supernatant for follow-up experiments.
+To demonstrate the mineralization capacity of LRAP, the supernatant was added into artificial saliva(8.7mM KCl,0.6mM MgCl<sub>2</sub>,10.mM CaCl<sub>2</sub>,4.6mM K<sub>2</sub>HPO<sub>4</sub>,2.7mM KH<sub>2</sub>PO<sub>4</sub>) to make the final protein concentration was 5mg/ml. The solution was incubated at 37℃ for 24h and the turbidity was observed(<b>Figure 3</b>)We observed that the +IPTG group had less precipitation and higher turbidity and we speculated the phenomenon is because that the formation of precipitation is prevented by the nucleation of LRAP and ions in artificial saliva.
+<img src=https://2020.igem.org/wiki/images/9/9d/T--HZAU-China--LRAPF3.jpg width=100% alt="">
+<br>
+<b>Figure 3.</b>Effects of different supernatants on turbidity of artificial Saliva.
+<br>
+We incubated the supernatant of ultrasound-broken bacteria which include LRAP together with pig teeth. Then we designed the experiment called “Toothtern-blot” to verify the existence and function of LRAP.
+The teeth of domestic pigs were broken into fragments about 5mm long on each side, which  were selected and stored in 75% alcohol at room temperature.
+The total protein concentration of the three supernatants was adjusted into 10mg/mL. Three pieces of pig teeth were washed with PBS buffer to remove the surface’s alcohol, then put in three 2mL centrifuge tubes respectively. After that, three supernatants with uniform protein concentration were added respectively. Incubate at 37℃ for 20h, and the tubes were upside  down for several times every 5h to eliminate the influence of protein deposition processes as much as possible.
+The incubated pig teeth fragments were repeatedly washed with PBS buffer for 2-3min to remove the protein that was not bound to the teeth.The rinsed pig teeth were incubated in plastic petri dishes with diameter of 10cm, diluted with 50 mL 5% skimmed milk at 1:10000 and hybridized with His-Tag, Rabbit pAb (YEASEN Biotech,Shanghai, China) at room temperature for 2h. Then  washed them with TBST buffer 3 times for 10min each. After that, diluted with 50 mL 5% skimmed milk at 1:10000 and hybridized with peroxidase-conjugated Goat anti-rabbit IgG (H+L) (YEASEN Biotech,Shanghai, China) at room temperature for 2h. Then washed them with TBST buffer 3 times for 10min each. Use A Super ECL Detection Reagent ECL (YEASEN Biotech,Shanghai, China)for development operations.
+The results showed that LRAP was able to bind to the tooth fragments and had binded more in the dentin(<b>Figure 4</b>).
+<br>
+<img src=https://2020.igem.org/wiki/images/c/c4/T--HZAU-China--LRAPF4.jpg width=100% alt="">
+<br>
+<b>Figure 4.</b>”Toothtern-blot” analysis of different supernatants.  <b>A and B . </b>The same result in different exposures.  <b>C. </b>Picture of the teeth in reality.