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	  <ul>
      <li><a href="#top">Header</a></li>
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	      <a href="#food">Details</a>
	      <ul>
	        <li><a href="#A1">Signal peptide</a></li>
	        <li><a href="#A2">The induction concentration of uric acid</a></li>
	        <li><a href="#A3">Express, urification and analysis of urate oxidase</a></li>
	        <li><a href="#A4">Cell penetrating peptide experiment</a></li>
                <li><a href="#A5">Colony number regulation experiment</a></li>
                <li><a href="#A6">Invitro lethal experiment</a></li>
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<img src="https://static.igem.org/mediawiki/2018/0/0b/T--DLUT_China--dna%E7%94%B5%E6%B3%B3.png" style="position:absolute;z-index:-1;left:650px;width:283px;height:370px;top:200px;"/>
<p   style="position:absolute;left:950px;top:300px; 
    writing-mode: vertical-lr;/*从左向右 从右向左是 writing-mode: vertical-rl;*/  
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    font-family: 'Cinzel', serif;color:purple;font-size:30px;">Birth of uox</p>
<a href="https://2018.igem.org/Team:DLUT_China/Experiments" style="position:fixed;top:580px;font-size:30px;left:0px;font-family: 'Dancing Script', cursive;">Experiments</a>
  <header id="top" class="responsive-fonts">
	<h1 style="font-size:40px;font-family: 'Cinzel', serif;position:absolute;top:400px;">Results</h1>
	
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		<article id="A1"><span style="font-size:30px;font-family: 'Cinzel', serif;color:purple;">1. Signal peptide</span><br><br><div style="font-size:20px;text-align:justify;width:800px;font-family: 'Oxygen', sans-serif;line-height:30px;">
<span style="font-size:25px;color:purple;">1.1 Plasmid transformation</span><br><br>
For the purpose of delivering the expressed urate oxidase into the blood, four secretion signal peptide sequences (PhoA ,which is an exocrine signal of the outer membrane protein alkaline phosphatase of E. coli; OpmA, which is an exocrine signal of the outer membrane protein of E. coli; STiI, which is an exocrine signal of the outer membrane protein heat stable enterotoxin II Stb of E. coli; PeLB, which can directs the protein to the bacterial periplasm), and four cell penetrating peptide sequences (DNP, which is a cyclic heptapeptide DNP that can be used as a permeation signal for Caco-2 cells; TAT peptide, which is derived from the transactivator of transcription of human immunodeficiency virus and is a cell-penetrating peptides; R8 (Octaarginine) , which is a representative cell-penetrating peptide which is a synthetic sequence; Pep-1, called HIV-reverse transcriptase/SV40 T-antigen) were chosen and fused expression with the target gene, respectively, to evaluate their ability.
<br><br>
Five DNA fragments we designed and constructed, which include four signal peptide with four cell penetrating peptidefused GFP encoded genes and urate oxidase encoded gene, were inserted into the multiple cloning site of the pET-22b plasmid, and then were introduced into the BL21 strain, and were selected on the basis of Amp resistance . To ensure that we introduced the plasmid into the BL21 strain, we performed colony PCR on colonies grown on solid media.      
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<center><img src="https://static.igem.org/mediawiki/2018/2/22/T--DLUT_China--resultsf1.png" style="width:60%;"/><br></center>
<center><h5>Figure 1  Results of agarose gel electrophoresis of colony PCR<br>
M: Marker DL2000; O: OpmA signal peptide sequence; P: PeLB signal peptide sequence; A: phoA signal peptide sequence; U: urate oxidase sequence; S: STiI signal peptide sequence.
</h5></center><br>
<center><img src="https://static.igem.org/mediawiki/2018/2/26/T--DLUT_China--results112.png" style="width:70%;"/><br></center>
<center><h5>Figure 2  Results of agarose gel electrophoresis of enzyme digestion<br>
DL: DL2000 marker;
PCR: A fragment obtained by PCR of a urate oxidase-encoding gene; 
PD: A target fragment obtained by PCR of a urate oxidase-encoding gene and performing enzyme digestion;
A: PhoA's target fragment;
AD: Target fragment of the phoA after digestion;
O: OpmA's target fragment;
OD: Target fragment of the OpmA after digestion;
B: PoLB's target fragment;
BD: Target fragment of the PoLB after digestion;
I: STiI's target fragment;
ID: Target fragment of the STiI after digestion;
ER: λEcoT14.

</h5></center><br>
<br>

<span style="font-size:25px;color:purple;">1.2 Testing Signal Peptide</span><br><br>
<center><h5 style="position:relative;bottom:-30px;"> Table 1  Group Description</h5></center>
<center><img src="https://static.igem.org/mediawiki/2018/2/22/T--DLUT_China--results121.png" style="width:80%"/><br>
<div style="font-size:20px;text-align:justify;width:800px;font-family: 'Oxygen', sans-serif;line-height:30px;">
The recombinant molecules were expressed induced by IPTG. The supernatant were collected to measure the absorbance at 525 nm using a wavelength of 395 nm in a microplate reader. By one-way analysis of variance (Table 2) and multiple comparisons (Table 3/Figure 3), the GFP fluorescence intensity of the four experimental groups was significantly different from the blank control, but the difference between the four signal peptides was not significant. So we concluded that the four signal peptides all function, but there is no one more effective.
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<center><h5>Table 2   ANOVA of four signal peptide functions</h5></center>
<center><img src="https://static.igem.org/mediawiki/2018/c/c7/T--DLUT_China--Table_1.png" style="width:554px;height:110px"/></center> <br>
<center><h5>Table 3   Significant difference of five group</h5></center>
<center><img src="https://static.igem.org/mediawiki/2018/7/70/T--DLUT_China--Table_2.png" style="width:316px;height:142px" /> </center><br>

<center><img src="https://static.igem.org/mediawiki/2018/d/d9/T--DLUT_China--Figure_2_Significant_difference_of_five_group.png" style="width:446px;height:277px;" /></center> 
<center><h5>Figure 3  Significant difference of five group</h5></center>
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</div></article>
		<article id="A2"><span style="font-size:30px;font-family: 'Cinzel', serif;color:purple;">2. The induction concentration of uric acid</span><br><br>
<div style="font-size:20px;text-align:justify;width:800px;font-family: 'Oxygen', sans-serif;line-height:30px;">



The feasibility of our HucR sequence (Plasmids from the Institute of Microbiology, Chinese Academy of Sciences, Tang Yan double group) . was verified using RFP (red fluorescent protein) as a reporter gene. When uric acid is absent or not high, RFP is not expressed; when uric acid concentration is sufficient, RFP is expressed and a red fluorescence is detetable. The pSHYa plasmid， which is kindly offered byProf. Tang, at the Institute of Microbiology, Chinese Academy of Sciences, has the HucR and huco coding sequences we need. We transformed the plasmid into the BL21 strain and performed relevant experiments. The experimental results are shown in the figures below.<br>
<img src="https://static.igem.org/mediawiki/2018/8/8c/T--DLUT_China--resf1.png"  /><br>
<center><img src="https://static.igem.org/mediawiki/2018/b/bc/T--DLUT_China--resf2.png"  style="width:70%;"  /><br></center>

From the experimental results, it was found that the control group without uric acid had no red fluorescence, and the experimental group with uric acid concentration at 10^–6 mol/L and uric acid concentration of 10^–5 mol/L shouwed only a small amount of red fluorescence. A large amount of red fluorescence was observed in the experimental group uric acid concentration at 10^–4 mol/L and  10^–3 mol/L. Therefore,  the optimum uric acid induction concentration was 10^–4 mol/L.
</div></article>
		<article id="A3"><span style="font-size:30px;font-family: 'Cinzel', serif;color:purple;width:600px;">3. Express, urification and analysis of urate oxidase</span><br><br>

<div style="font-size:20px;text-align:justify;width:800px;font-family: 'Oxygen', sans-serif;line-height:30px;">
<span style="font-size:25px;color:purple;">3.1 Express urate oxidase</span><br><br>
A plasmid containing a urate oxidase sequence was introduced into the BL21 strain, and IPTG was used as an inducer to induce expression of urate oxidase. After expression, the cells were disrupted by sonication and the supernatant was taken. The supernatant (extracellular protein) of the first centrifugation and the supernatant (intracellular protein) after sonication were subjected to polyacrylamide gel electrophoresis. The electrophoresis results are shown in Figure 8. <br>
<center><img src="https://static.igem.org/mediawiki/2018/c/c3/T--DLUT_China--99.png" style="width:439px;height:404px;"/></center>
<center><h5>Figure 4  Polyacrylamide gel electrophoresis results<br>
BP: Negative control (blank) precipitation; BS: Negative control (blank) supernatant; PD: Precipitation after disruption of cells;  SD: Supernatant after disruption of cells; M: Marker.
</h5></center><br>
The molecular weight of our urate oxidase is 35.10 KDa. From the electrophoresis results, the molecular weight of the protein molecule expressed by the recombinant strain is between 43.0KDa and 31.0KDa, which is consistent with the molecular weight of our urate oxidase. To prove that our urate oxidase was expressed. And there are bands in the precipitation and supernatant of the cell disrupting solution, which proves our expessed protein is partially soluble.<br><br>
<span style="font-size:25px;color:purple;">3.2 Urification and analysis of urate oxidase</span><br><br>
We used AKTA to purify our protein molecules and use western blot analysis (since our protein molecule has a 6xHis tag, we can perform purification and western blot analysis), the results are shown below.<br>
<center><img src="https://static.igem.org/mediawiki/2018/1/17/T--DLUT_China--100.png" style="width:490px;height:394px;"/></center>
<center><h5>Figure 5    Polyacrylamide gel electrophoresis results<br>
BP: Negative control (blank) precipitation; BS: Negative control (blank) supernatant; PD: Precipitation after disruption of cells;  SD: Supernatant after disruption of cells; M: Marker; FT: Flow through solution; 20mM: Imidazole eluate at a concentration of 20 mM/L; 50mM: Imidazole eluate at a concentration of 50 mM/L; 250mM: Imidazole eluate at a concentration of 250 mM/L; 500mM: Imidazole eluate at a concentration of 500 mM/L.

</h5></center><br>
From the results of the purification, the data we obtained is not ideal, and the big reason is that our operation is not standardized.
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</article>

   <article id="A4">
<span style="font-size:30px;font-family: 'Cinzel', serif;color:purple;">4. Cell penetrating peptide experiment</span><br><br><div style="font-size:20px;text-align:justify;width:800px;font-family: 'Oxygen', sans-serif;line-height:30px;">
<span style="font-size:25px;color:purple;">4.1	GFP Expression</span><br><br>
We set three IPTG concentrations (0.1 mM, 0.5 mM, 1.0 mM) and detected GFP with FBW under a fluorescence microscope. The ST11 experimental group showed the strongest fluorescence intensity. In other experimental groups, the fluorescence was weak, and the fluorescence of E. coli could not be observed in the blank control. Comparing different inducer concentrations, the difference between 3 groups was found to be insignificant. We chose an inducer concentration of 0.5 mM for subsequent experiments.<br><br>
<center><img src="https://static.igem.org/mediawiki/2018/8/8a/T--DLUT_China--Figure_3.png" style="width:100%;"/></center><br>
<center><h5>Figure 6  GFP fluorescence expression image<br>
A: Blank control without GFP, 20-fold fluorescence microscope;
B: Positive control group, 20-fold fluorescence microscope;
C: 2b2 experimental group with 0.1mM IPTG, 20-fold fluorescence microscope;
D: 2b2 experimental group with 0.5mM IPTG, 20-fold fluorescence microscope;
E: 2b2 experimental group with 1.0mM IPTG, 20-fold fluorescence microscope;
F: PolB experimental group with 0.1mM IPTG, 20-fold fluorescence microscope;
G: PolB experimental group with 0.5mM IPTG, 20-fold fluorescence microscope;
H: PolB experimental group with 1.0mM IPTG, 20-fold fluorescence microscope;
I: ST11 experimental group with 0.1mM IPTG, 20-fold fluorescence microscope;
J: ST11 experimental group with 0.5mM IPTG, 20-fold fluorescence microscope;
K: ST11 experimental group with 1.0mM IPTG, 20-fold fluorescence microscope;
L: HIV experimental group with 0.1mM IPTG, 20-fold fluorescence microscope;
M: HIV experimental group with 0.5mM IPTG, 20-fold fluorescence microscope;
N: HIV experimental group with 1.0mM IPTG, 20-fold fluorescence microscope.

</h5></center><br>
<span style="font-size:25px;color:purple;">4.2 Observing GFP in HeLa cells</span><br><br>
Fluorescence microscopy was used to detect intracellular GFP by FBW. It was found that each experimental group had weak green fluorescence, and there was no fluorescence in the blank group and the negative control group.<br>
<center><img src="https://static.igem.org/mediawiki/2018/3/38/T--DLUT_China--Figure_11.png" style="width:100%;"/></center><br>
<center><h5>Figure 7  Observing GFP in HeLa Cells<br>
A: Blank control, no GFP, 10-fold fluorescence microscope; 
B: Negative control, GFP without penetrating peptide, 10-fold fluorescence microscope; 
C: 2b2 experimental group, 10-fold fluorescence microscope;
D: ST11 experimental group, 10-fold fluorescence microscope;
E: PolB experimental group, 10-fold fluorescence microscope;
F: HIV experimental group, 10-fold fluorescence microscope. 


</h5></center><br>
<center><img src="https://static.igem.org/mediawiki/2018/1/1a/T--DLUT_China--Figure_12.png
" style="width:550px;height:339px;"/></center>
<center><h5>Figure 8  Results of SDS-PAGE to test GFP in HeLa cells<br></center><br>
<span style="font-size:25px;color:purple;">4.3	Results of SDS-PAGE to test GFP in HeLa cells</span><br><br>

The positive control P1, P2 are cells supernatant after disruption which contains GFP; the negative control is the inclusion of cells incubated with GFP which has no penetrating peptide. The blank control is the inclusion of cells incubated with DMEM medium. Comparing 4 penetrating peptide, it shows that R8 penetrating peptide and cyclic heptapeptide DNP work best.


</div>
</article>


		<article id="A5">
<span style="font-size:30px;font-family: 'Cinzel', serif;color:purple;">5. Colony number regulation experiment</span><br><br>
<div style="font-size:20px;text-align:justify;width:800px;font-family: 'Oxygen', sans-serif;line-height:30px;">
We adjusted the initial Abs600 values of the bacteria to the same value and measured the value of Abs600 every 30 minutes to obtain the following results.
<center><img src="https://static.igem.org/mediawiki/2018/thumb/2/2e/T--DLUT_China--GP2.png/800px-T--DLUT_China--GP2.png
" style="width:70%;"/></center>
<center><h5>Figure 9   Colony number regulation experiment result</h5></center>
<br>
It can be seen from the results that when the bacteria grow to Abs600 to about 0.25, the growth curve of the bacteria basically does not change, and the negative control group is still growing, indicating that our colony control system has achieved the expected effect.
</div></article>

		<article id="A6">
<span style="font-size:30px;font-family: 'Cinzel', serif;color:purple;">6. Invitro lethal experiment</span><br><br>
<div style="font-size:20px;text-align:justify;width:800px;font-family: 'Oxygen', sans-serif;line-height:30px;">
<center><img src="
 https://static.igem.org/mediawiki/2018/f/fd/T--DLUT_China--results51.png
" style="width:70%;"/></center>

HucO is regulated by HucR. When uric acid is abscent, HucR can bind to hucO making downstream gene silencing. Lysis1 and Lysis2 are lyases. Lysis1siRNA286 can inhibit Lysis1, and Lysis2siRNA106 can inhibit Lysis2.
When both plasmids are simultaneously expressed, the lyases are inhibited. But if any plasmid is lost or HucR combined hucO，part3 will not be expressed. And the bacterium is cleaved by the Lysis2 .<br>
A series of things happens when bacteria are excreted: First, the concentration of uric acid get lower. Second, the hucO of part3 is suppressed by HucR and cannot work. Next, Lysis2 siRNA106 cannot inhibit Lysis2. Finally, the bacteria are cleaved by Lysis2.<br>
To test the lethal ability of part2 and part3, and the regulation of uric acid on part3 we did four sets of experiments.<br>

Blank is a plasmid-free BL21 strain.<br>
Part3 is a BL21 strain containing the part3 plasmid.<br>
Part2 is a BL21 strain containing the part2 plasmid.<br>
Part3-with uric acid is a BL21 strain containing part3 plasmid cultured with a uric acid concentration of 10-4M.
<br>
<center>
<img src="https://static.igem.org/mediawiki/2018/2/2b/T--DLUT_China--results61.png" style="width:562px;height:257px;"/></center>
<center><h5>Figure 10   Colony number regulation experiment result

</h5></center>
<br>
We can get three conclusions:<br>
1.Part2 and part3 have a certain lethal effect, but they are need to be improved.<br>
2. It can be seen that in the first two hours, uric acid has a certain regulatory effect on part3.<br>
3. However, after 2 hours, there may be cases where uric acid is degraded, and further experimental verification is needed.
<br>
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