Difference between revisions of "Part:BBa K2644001"
 
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<partinfo>BBa_K2644001 short</partinfo>
 
<partinfo>BBa_K2644001 short</partinfo>
  
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A gene for fluorescent protein smURFP and enzyme HOI
+
A gene for fluorescent protein smURFP and the precursor of biliverdin--HO-I
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here
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<partinfo>BBa_K2644001 parameters</partinfo>
 
<partinfo>BBa_K2644001 parameters</partinfo>
 
<!-- -->
 
<!-- -->
 +
 +
===Usage===
 +
smURFP (small ultra-red FP) and HO-I is an important part in our group. It can experss smURFP along with the precursor of biliverdin--HO-I. As we know, smURFP has many advantages compared with other cyan, green, yellow and orange FPs, for it molecule less light is scattered, absorbed, or re-emitted by endogenous biomolecules. It has 642/670 nm excitation - emission peaks, a large extinction coefficient and quantum yield, and photostability comparable to that of eGFP. However, it must be combined with biliverdin (BV) to fluoresce. So in order to make it more convenient to observe fluorescence in the bacteria, we set up this composite part.
 +
 +
===Biology===
 +
It's obvious that smURFP can't fluoresce without BV. So we construct the co-expression system, where the gene of fluorescent protein---smURFP and the gene of HO-1 should be connected to the same expression vector and then transferred to our target bacteria. The HO-I 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. Then we can see activated light of 660nm (ultra-red light)through the laser confocal microscopy.
 +
 +
===Reference===
 +
[1] John V Frangioni. In vivo near-infrared fluorescence imaging. [J].Current Opinion in Chemical Biology 2003, 7:626–634
 +
[2] 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 constructed the part smURFP+HOI and did some experiments on it.
 +
 +
===PCR and enzyme digestion===
 +
First, we used PCR to amplify the senquence for smURFP+HO-I. We did point mutation on it so that it could be digested by restriction enzymes EcoRI and PstI.
 +
 +
  https://static.igem.org/mediawiki/2018/b/bb/T--TJU_China--1-1.png<br>
 +
'''Figure 1.'''Left fragment PCR for point mutation(length:428bp). Using marker 2000.
 +
<br>
 +
 +
  https://static.igem.org/mediawiki/2018/7/75/T--TJU_China--1-2.png<br>
 +
'''Figure 2.'''Right fragment PCR for point mutation(length:783bp). Using marker 2000.
 +
<br>
 +
 +
  https://static.igem.org/mediawiki/2018/3/30/T--TJU_China--1-3.png<br>
 +
'''Figure 3.'''Result of point mutation(length:1217bp). Using marker 2000.
 +
<br>
 +
 +
  https://static.igem.org/mediawiki/2018/7/77/T--TJU_China--1-4.png<br>
 +
'''Figure 4.'''Result of enzyme digestion. Using marker 2000.
 +
<br>
 +
 +
===Connection, transformation and plasmid DNA extraction===
 +
We used T4 DNA Ligase to connect smURFP+HO-I with standardized plasmid(2000bp) in order to transform it into E.coli DH5α.After culturing for a few days, we could get recombinant plasmids.
 +
 +
  https://static.igem.org/mediawiki/2018/f/f2/T--TJU_China--1-5.png<br>
 +
'''Figure 5.'''Result of plasmid DNA extraction. Using marker 2000 plus.(a)10 μl sample of original recombinant plasmid. (b)10 μl sample of senquence digested by restriction enzyme EcoRI. (c)10 μl sample of senquence digested by restriction enzyme PstI. (d)10 μl sample of senquence digested by restriction enzymes EcoRI and PstI.
 +
<br>
 +
 +
===Protein production and purification===
 +
 +
After the expression of protein by E.coli BL21, the products were purified for several times. Figure 6 reflects the condition of sample after purification of the nickel column. The first is before purification, and 7 and 8 were purified ones.
 +
 +
    https://static.igem.org/mediawiki/parts/6/68/Niezhu.png<br>
 +
'''Figure 6.'''  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.
 +
 +
<br>
 +
 +
 +
We then used the AKTA system and molecular sieve system for further purification.
 +
 +
    https://static.igem.org/mediawiki/parts/thumb/a/ac/AKTA.png/800px-AKTA.png<br>
 +
'''Figure 7.'''  Result of purification of AKTA system.<br>
 +
 +
    https://static.igem.org/mediawiki/parts/thumb/3/3d/MSS.png/800px-MSS.png<br>
 +
'''Figure 8.'''  Result of purification of molecular sieve system.<br>
 +
 +
 +
    https://static.igem.org/mediawiki/parts/a/a5/AKTAMSS.png<br>
 +
'''Figure 9.'''  . 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.
 +
<br>
 +
 +
 +
----
 +
 +
===Protein crystallization===
 +
 +
    https://static.igem.org/mediawiki/parts/c/c6/Cristal.png<br>
 +
'''Figure 10.'''  Result of proper condition for crystallization.<br>
 +
 +
 +
----
 +
 +
===Co-expression with HO-1 in E.coli BL21 (<i>in vitro</i>)===
 +
 +
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;">
 +
    https://static.igem.org/mediawiki/parts/e/e5/Microplate_Reader.png<br>
 +
'''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>
 +
</p>
 +
 +
 +
 +
Then laser confocal microscopy was use to observe these bacteria, activate light of 640nm was used, as shown in Figure 11.
 +
 +
<p style="text-align: center;">
 +
    https://static.igem.org/mediawiki/2018/4/46/T--TJU_China--1-7.png<br>
 +
 +
  https://static.igem.org/mediawiki/2018/b/b9/T--TJU_China--1-6.png<br>
 +
'''Figure 11.'''  The result after induction, the upper one is the control group, and the inferior one is the experimental group.<br>
 +
</p>

Latest revision as of 04:15, 16 October 2018


smURFP+HOI

A gene for fluorescent protein smURFP and the precursor of biliverdin--HO-I

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) and HO-I is an important part in our group. It can experss smURFP along with the precursor of biliverdin--HO-I. As we know, smURFP has many advantages compared with other cyan, green, yellow and orange FPs, for it molecule less light is scattered, absorbed, or re-emitted by endogenous biomolecules. It has 642/670 nm excitation - emission peaks, a large extinction coefficient and quantum yield, and photostability comparable to that of eGFP. However, it must be combined with biliverdin (BV) to fluoresce. So in order to make it more convenient to observe fluorescence in the bacteria, we set up this composite part.

Biology

It's obvious that smURFP can't fluoresce without BV. So we construct the co-expression system, where the gene of fluorescent protein---smURFP and the gene of HO-1 should be connected to the same expression vector and then transferred to our target bacteria. The HO-I 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. Then we can see activated light of 660nm (ultra-red light)through the laser confocal microscopy.

Reference

[1] John V Frangioni. In vivo near-infrared fluorescence imaging. [J].Current Opinion in Chemical Biology 2003, 7:626–634 [2] 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 constructed the part smURFP+HOI and did some experiments on it.

PCR and enzyme digestion

First, we used PCR to amplify the senquence for smURFP+HO-I. We did point mutation on it so that it could be digested by restriction enzymes EcoRI and PstI. 

  T--TJU_China--1-1.png

Figure 1.Left fragment PCR for point mutation(length:428bp). Using marker 2000. 

  T--TJU_China--1-2.png

Figure 2.Right fragment PCR for point mutation(length:783bp). Using marker 2000. 

  T--TJU_China--1-3.png

Figure 3.Result of point mutation(length:1217bp). Using marker 2000. 

  T--TJU_China--1-4.png

Figure 4.Result of enzyme digestion. Using marker 2000.

Connection, transformation and plasmid DNA extraction

We used T4 DNA Ligase to connect smURFP+HO-I with standardized plasmid(2000bp) in order to transform it into E.coli DH5α.After culturing for a few days, we could get recombinant plasmids. 

  T--TJU_China--1-5.png

Figure 5.Result of plasmid DNA extraction. Using marker 2000 plus.(a)10 μl sample of original recombinant plasmid. (b)10 μl sample of senquence digested by restriction enzyme EcoRI. (c)10 μl sample of senquence digested by restriction enzyme PstI. (d)10 μl sample of senquence digested by restriction enzymes EcoRI and PstI.

Protein production and purification

After the expression of protein by E.coli BL21, the products were purified for several times. Figure 6 reflects the condition of sample after purification of the nickel column. The first is before purification, and 7 and 8 were purified ones. 

   Niezhu.png

Figure 6. 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.



We then used the AKTA system and molecular sieve system for further purification. 

   800px-AKTA.png

Figure 7. Result of purification of AKTA system.

   800px-MSS.png

Figure 8. Result of purification of molecular sieve system.


   AKTAMSS.png

Figure 9. . 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.


Protein crystallization

   Cristal.png

Figure 10. Result of proper condition for crystallization.


Co-expression with HO-1 in E.coli BL21 (in vitro)

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.

Microplate_Reader.png
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.


Then laser confocal microscopy was use to observe these bacteria, activate light of 640nm was used, as shown in Figure 11.

T--TJU_China--1-7.png
T--TJU_China--1-6.png
Figure 11. The result after induction, the upper one is the control group, and the inferior one is the experimental group.