Difference between revisions of "Part:BBa K4115044"

(Sequence and Features)
 
(27 intermediate revisions by the same user not shown)
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|-
 
|-
 
|'''Function'''
 
|'''Function'''
|gene reporter
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|Gene reporter
 
|-
 
|-
 
|'''Use in'''
 
|'''Use in'''
|E.coli
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|<i>E. coli</i>
 
|-
 
|-
 
|'''RFC standard'''
 
|'''RFC standard'''
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|-
 
|-
 
|'''Submitted by'''
 
|'''Submitted by'''
|[https://2022.igem.wiki/shanghaitech-china/ (ShanghaiTech_China)]
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|[https://2022.igem.wiki/shanghaitech-china/ ShanghaiTech_China]
 
|}
 
|}
  
This composite part is used to express the tagRFP[https://parts.igem.org/Part:BBa_K4115013 (BBa_K4115013)] by a constitutive promoter J23101[https://parts.igem.org/Part:BBa_J23101 (J23101)], thus we can use red fluorescence as a detection.
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This composite part is used to express the tagRFP[https://parts.igem.org/Part:BBa_K4115013 (BBa_K4115013)] by a constitutive promoter J23101[https://parts.igem.org/Part:BBa_J23101 (BBa_J23101)], thus we can use red fluorescence as a detection.<br><br>
 +
We first transformed this composite part into the plasmid backbone PUC57 to verify that it could express tagRFP protein. Later, we will transform it into the plasmid backbone pBBR1 and transfer it into <i>A.caulinodans</i> ORS571 as a shuttle plasmid to mark <i>A.caulinodans</i> ORS571.
  
  
 
===Experimental approach===
 
===Experimental approach===
  
To wrap the e.coli into the alginate gel beads, we first weigh 4g of sodium alginate, add 100ml of hot water, mix well, put in an autoclave, 1.0kg/cm sterilization for 10min, stand at room temperature for 2h, until the bubbles disappear. Then, Take 10 g of E. coli with tagRFP transformed wet cells, suspend them in 10 ml of sterile physiological saline, and prepare a bacterial suspension. After that, we slowly add the above sodium alginate solution and stir to make a suspension. The above suspension was gradually dropped into 1000ml of 0.2mol/LCaCL2 solution with a syringe with a 9-gauge needle and stirred with an electromagnetic stirrer while injecting to form bead-like particles (diameter 3ram). Finally, we soak in CaCL2 solution for 2h, then weigh 4g of sodium alginate and add 100ml of hot water, mix well, put in a high-pressure sterilizer, 1.0kg/cm sterilization for 10min, stand at room temperature for 2h, until gas retention disappears.
+
To wrap the <i>E. coli</i> into the alginate gel beads, we first weigh 4g of sodium alginate, add 100ml of hot water, mix well, put in an autoclave, 1.0kg/cm sterilization for 10min, stand at room temperature for 2h, until the bubbles disappear. Then, Take 10 g of E. coli with tagRFP transformed wet cells, suspend them in 10 ml of sterile physiological saline, and prepare a bacterial suspension. After that, we slowly add the above sodium alginate solution and stir to make a suspension. The above suspension was gradually dropped into 1000ml of 0.2mol/LCaCL2 solution with a syringe with a 9-gauge needle and stirred with an electromagnetic stirrer while injecting to form bead-like particles (diameter 3ram). Finally, we soak in CaCL2 solution for 2h, then weigh 4g of sodium alginate and add 100ml of hot water, mix well, put in a high-pressure sterilizer, 1.0kg/cm sterilization for 10min, stand at room temperature for 2h, until gas retention disappears.
  
==Proof of function==
+
==Proof of express==
  
After We transformed this plasmid into E. coli and encapsulated the E. coli into alginate gel beads, we then induced it to express tagRFP. We photograph the cross-section of the gel beads under a confocal microscope and get Figure 1.
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After We transformed this plasmid into E. coli and encapsulated the E. coli into alginate gel beads, we then induced it to express tagRFP. We photograph the cross-section of the gel beads under a confocal microscope and get Figure 1 and Figure 2.
  
[[Image:TagRFP in gel beads.jpg|700px|thumb|left|'''Figure 1:''' Confocal image of cross-section of gel beads with red fluorescent protein E.coli.]]
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[[File:TagRFP in gel beads 10X.tif|430px|thumb|left|'''Figure 1:''' 10X Confocal image of cross-section of gel beads with red fluorescent protein <i>E. coli</i>.]]
  
 +
[[File:TagRFP in gel beads 63X.tif|430px|thumb|right|'''Figure 2:''' 63X Confocal image of cross-section of gel beads with red fluorescent protein <i>E. coli</i>.]]<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br>
  
 
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<!-- Add more about the biology of this part here
<!-- Add more about the biology of this part here>
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===Usage and Biology===
 
===Usage and Biology===
  
 
<!-- -->
 
<!-- -->
<span class='h3bb'>
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<span class='h3bb'>Sequence and Features</span>
 
+
<partinfo>BBa_K4115039 SequenceAndFeatures</partinfo>
 
+
 
+
 
+
==Sequence and Features==
+
</span>
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+
<partinfo>BBa_K4115044 SequenceAndFeatures</partinfo>
+
  
 
== Reference ==
 
== Reference ==

Latest revision as of 02:47, 5 October 2022


J23101-B0034-tagRFP-B0015

J23101-B0034-tagRFP-B0015
Function Gene reporter
Use in E. coli
RFC standard RFC 10, RFC 10 compatible
Backbone pUC, pBBR1
Submitted by ShanghaiTech_China

This composite part is used to express the tagRFP(BBa_K4115013) by a constitutive promoter J23101(BBa_J23101), thus we can use red fluorescence as a detection.

We first transformed this composite part into the plasmid backbone PUC57 to verify that it could express tagRFP protein. Later, we will transform it into the plasmid backbone pBBR1 and transfer it into A.caulinodans ORS571 as a shuttle plasmid to mark A.caulinodans ORS571.


Experimental approach

To wrap the E. coli into the alginate gel beads, we first weigh 4g of sodium alginate, add 100ml of hot water, mix well, put in an autoclave, 1.0kg/cm sterilization for 10min, stand at room temperature for 2h, until the bubbles disappear. Then, Take 10 g of E. coli with tagRFP transformed wet cells, suspend them in 10 ml of sterile physiological saline, and prepare a bacterial suspension. After that, we slowly add the above sodium alginate solution and stir to make a suspension. The above suspension was gradually dropped into 1000ml of 0.2mol/LCaCL2 solution with a syringe with a 9-gauge needle and stirred with an electromagnetic stirrer while injecting to form bead-like particles (diameter 3ram). Finally, we soak in CaCL2 solution for 2h, then weigh 4g of sodium alginate and add 100ml of hot water, mix well, put in a high-pressure sterilizer, 1.0kg/cm sterilization for 10min, stand at room temperature for 2h, until gas retention disappears.

Proof of express

After We transformed this plasmid into E. coli and encapsulated the E. coli into alginate gel beads, we then induced it to express tagRFP. We photograph the cross-section of the gel beads under a confocal microscope and get Figure 1 and Figure 2.

Figure 1: 10X Confocal image of cross-section of gel beads with red fluorescent protein E. coli.
Figure 2: 63X Confocal image of cross-section of gel beads with red fluorescent protein E. coli.






















Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 985

Reference

[1] 赵景联.固定化大肠杆菌细胞生产γ-氨基丁酸的研究[J].生物工程学报,1989,5(02):124-128.DOI:10.13345/j.cjb.1989.02.010.