Difference between revisions of "Part:BBa K2328000"

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===Usage===
 
===Usage===
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smURFP (small ultra-red FP) is an important part in our group. It is desirable for our in-vivo imaging because with it molecule less light is scattered, absorbed, or re-emitted by endogenous biomolecules compared with cyan, green, yellow and orange FPs. smURFP can covalently attaches a biliverdin(BV) chromophore without a lyase, and has 642/670 nm excitation - emission peaks, a large extinction coefficient and quantum yield, and photostability comparable to that of eGFP.
+
smURFP (small ultra-red FP) is an important part in our group. It is desirable for our BV detection and in-vivo imaging because with it molecule less light is scattered, absorbed, or re-emitted by endogenous biomolecules compared with cyan, green, yellow and orange FPs. smURFP can covalently attaches a biliverdin(BV) chromophore without a lyase, and has 642/670 nm excitation - emission peaks, a large extinction coefficient and quantum yield, and photostability comparable to that of eGFP.
  
 
===Biology===
 
===Biology===
−
In order to fluoresce, smURFP must be combined with biliverdin (BV) .We have two solutions to make in-vivo imaging come true. The first one is co-expression system and the other one is surface display system. To construct the co-expression system, the gene of fluorescent protein---smURFP and the gene of the precursor of biliverdin---HO-1 should be connected to the same expression vector and then transferred to our target bacteria. The precursor of biliverdin will be transferred to biliverdin through a series of conversion, and then fluorescent protein will combine with biliverdin directly in our target bacteria and glow in the bacteria. To construct the surface display system, the gene of fluorescent protein---smURFP and the gene of the anchoring protein should be connected to the same expression vector. After the recombinant plasmid is transferred to the target bacteria, the fluorescent protein and anchoring protein will express at the same time and become fusion protein, and then the fluorescent protein will be carried to the cell surface by anchoring protein. With the added biliverdin, fluorescent protein will combine with biliverdin and glow on the cell surface.
+
In order to fluorescence, smURFP must be combined with biliverdin (BV) .So we construct the surface display system to make in-vivo imaging come true. To construct the surface display system, the gene of fluorescent protein---smURFP and the gene of the anchoring protein should be connected to the same expression vector. After the recombinant plasmid is transferred to the target bacteria, the fluorescent protein and anchoring protein will express at the same time and become fusion protein, and then the fluorescent protein will be carried to the cell surface by anchoring protein. With the added biliverdin, fluorescent protein will combine with biliverdin and glow on the cell surface.
  
 
===Reference===
 
===Reference===
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==Results==
 
==Results==
−
We did many experiments on smURFP.  
+
We did some experiments on smURFP and BV.  
  
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===Protein production and purification===
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===Protein and BV===
  
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After the expression of protein by E.coli BL21, the products were purified for several times. Figure 1 reflects the condition of sample after purification of the nickel column. The first is before purification, and 2 and 3 were purified ones.  
+
We test the fluorescence changes of different concentration of smURFP and BV and the fluorescence.
  
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    https://static.igem.org/mediawiki/parts/6/68/Niezhu.png<br>
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https://static.igem.org/mediawiki/parts/thumb/7/7b/234.png/800px-234.png<br>
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'''Figure 1.'''  To test the purification efficiency and condition of Nickel Column Purification is via SDS-PAGE method. (a) The marker. (b) The 20 μl sample of E.coli crushing fluid before Nickel Column Purification with the target part of smURFP and his-sumo tag, around 26 kD. (c) The 20 μl sample of the solution with the target part is after Nickel Column Purification. (d)The 20 μl sample of the solution after is Nickel Column Purification is before the sumo protease digestion, with sumo protease around 13 kD. (e) The 20 μl sample of the Nickel medium is after 12 h sumo protease digestion with the target protein smURFP, around 12 kD. (f) The 20 μl sample of the eluent is with the protein smURFP. (g) The 20 μl sample of the Nickel medium after the elution to test whether the target protein is all in the solution.
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'''Figure 1.'''  A standard curve of fluorescence intensity changes of different concentration of smURFP and BV.<br>
  
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<br>
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===Surface display system in E.coli BL21 (<i>in vitro</i>)===
  
  
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We then used the AKTA system and molecular sieve system for further purification.  
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The laser confocal microscopy was use to observe these bacteria, activate light of 640nm was used, as shown in Figure 2.
  
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    https://static.igem.org/mediawiki/parts/thumb/a/ac/AKTA.png/800px-AKTA.png<br>
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<p style="text-align: center;">
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'''Figure 2.'''  Result of purification of AKTA system.<br>
+
     https://static.igem.org/mediawiki/parts/f/f1/Confocal.jpg<br>
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+
'''Figure 2.'''  The result after induction, as we can see, fluorescent protein combine with biliverdin and glow on the cell surface.
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    https://static.igem.org/mediawiki/parts/thumb/3/3d/MSS.png/800px-MSS.png<br>
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'''Figure 3.'''  Result of purification of molecular sieve system.<br>
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+
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+
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     https://static.igem.org/mediawiki/parts/a/a5/AKTAMSS.png<br>
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'''Figure 4.'''  . To test the purification efficiency and condition of AKTA Ion Exchange Chromatography and Molecular Sieve Purification. (a) The 20 μl sample of the solution before Ion Exchange Chromatography Purification is as a compare. (b)~(h) The 20 μl sample of the solution is after Ion Exchange Chromatography Purification. (i) The marker. (j) The 20 μl sample of the solution before Molecular Sieve Purification is as a compare. (l)~(o) The 20 μl sample of the solution is after Molecular Sieve Purification.
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<br>
+
  
  
 
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 +
===Surface display system in E.coli BL21 (<i>in vivo</i>)===
  
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===Protein crystallization===
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After tests <i>in vitro</i>, we used this engineered bacteria for experiments <i>in vivo</i>.. Utilizing Animal imaging system, we consistently observed the fluorescence emitted from the bacteria in mices' gut. The result successfully showed that our system was executable and excellent. And smURFP has very competible persistence and penetrability.
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+
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    https://static.igem.org/mediawiki/parts/c/c6/Cristal.png<br>
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'''Figure 5.'''  Result of proper condition for crystallization.<br>
+
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+
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+
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----
+
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+
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===Protein and BV===
+
−
 
+
−
We test the fluorescence changes of different concentration of smURFP and BV and the fluorescence.
+
−
 
+
−
https://static.igem.org/mediawiki/parts/7/75/Curve.png<br>
+
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'''Figure 6.'''  A series of curves of fluorescence intensity changes of different concentration of smURFP and BV.<br>
+
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+
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+
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----
+
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+
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===Co-expression with HO-1 in E.coli BL21===
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Plasmid pET28b with smURFP and HO-1 gene were transformed into E. coli BL-21. We used this induced bacteria to confirm the fluorescence and data showed a relatively high value, as shown in table 1.  
+
  
 
<p style="text-align: center;">
 
<p style="text-align: center;">
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     https://static.igem.org/mediawiki/parts/e/e5/Microplate_Reader.png<br>
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     https://static.igem.org/mediawiki/2017/8/89/INVIVO.png<br>
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'''Table 1.'''  Result of Microplate Reader in the black 96-well plate. Tube 1 and 2 are experimental group, and tube 3 is the control group.<br>
+
'''Figure 3.'''  The fluorescent inensity after doing intragastric administration for 5.5h.The left is the control one, the right is the experimental one.<br>
 
</p>
 
</p>
  
  
−
 
+
After the experiment, we gathered the data of RFU and drawed a gragh about its trend.  
−
Then laser confocal microscopy was use to observe these bacteria, activate light of 640nm was used, as shown in Figure 7.
+
  
 
<p style="text-align: center;">
 
<p style="text-align: center;">
−
     https://static.igem.org/mediawiki/parts/f/f1/Confocal.jpg<br>
+
     https://static.igem.org/mediawiki/parts/f/fe/Invivo.png<br>
−
'''Figure 7.'''  The result after induction, the upper one is the control group, and the inferior one is the experimental group.<br>
+
'''Figure 4.'''  Trend of RFU in vivo. M2 is the mice with 10^8 CFU bacteria, and M3 is the mice with 10^11 CFU. We can see the fluorescence is relatively high, and the RFU is still over 3000 after 300min when the mice with 10^11 CFU bacteria.<br>
 
</p>
 
</p>
  
 
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Latest revision as of 04:21, 16 October 2018


smURFP (I, codon-optimized for Escherichia coli) (without terminator codon TAA)

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
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Usage

smURFP (small ultra-red FP) is an important part in our group. It is desirable for our BV detection and in-vivo imaging because with it molecule less light is scattered, absorbed, or re-emitted by endogenous biomolecules compared with cyan, green, yellow and orange FPs. smURFP can covalently attaches a biliverdin(BV) chromophore without a lyase, and has 642/670 nm excitation - emission peaks, a large extinction coefficient and quantum yield, and photostability comparable to that of eGFP.

Biology

In order to fluorescence, smURFP must be combined with biliverdin (BV) .So we construct the surface display system to make in-vivo imaging come true. To construct the surface display system, the gene of fluorescent protein---smURFP and the gene of the anchoring protein should be connected to the same expression vector. After the recombinant plasmid is transferred to the target bacteria, the fluorescent protein and anchoring protein will express at the same time and become fusion protein, and then the fluorescent protein will be carried to the cell surface by anchoring protein. With the added biliverdin, fluorescent protein will combine with biliverdin and glow on the cell surface.

Reference

[1] Rodriguez EA,Tran GN , Gross LA, et al. A far-red fluorescent protein evolved from a cyanobacterial phycobiliprotein .[J].NATURE METHODS,2016:763-769.

Results

We did some experiments on smURFP and BV.

Protein and BV

We test the fluorescence changes of different concentration of smURFP and BV and the fluorescence. 

800px-234.png

Figure 1. A standard curve of fluorescence intensity changes of different concentration of smURFP and BV.

Surface display system in E.coli BL21 (in vitro)

The laser confocal microscopy was use to observe these bacteria, activate light of 640nm was used, as shown in Figure 2.

Confocal.jpg
Figure 2. The result after induction, as we can see, fluorescent protein combine with biliverdin and glow on the cell surface.

Surface display system in E.coli BL21 (in vivo)

After tests in vitro, we used this engineered bacteria for experiments in vivo.. Utilizing Animal imaging system, we consistently observed the fluorescence emitted from the bacteria in mices' gut. The result successfully showed that our system was executable and excellent. And smURFP has very competible persistence and penetrability.

<p style="text-align: center;"> 

   INVIVO.png

Figure 3. The fluorescent inensity after doing intragastric administration for 5.5h.The left is the control one, the right is the experimental one.


After the experiment, we gathered the data of RFU and drawed a gragh about its trend.

Invivo.png
Figure 4. Trend of RFU in vivo. M2 is the mice with 10^8 CFU bacteria, and M3 is the mice with 10^11 CFU. We can see the fluorescence is relatively high, and the RFU is still over 3000 after 300min when the mice with 10^11 CFU bacteria.