Difference between revisions of "Part:BBa K3716012"

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# Add 100 µl bacteria culture medium into a sterile 96-well plate. Measure fluorescence at 488nm excitation and 502nm absorption, and then also OD600 for normalization, with a microplate reader.
 
# Add 100 µl bacteria culture medium into a sterile 96-well plate. Measure fluorescence at 488nm excitation and 502nm absorption, and then also OD600 for normalization, with a microplate reader.
 
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# SDS-PAGE. The cells are lysed by 150 μL 4% SDS for 5 min at room temperature, then 10 min at 95℃. Then 6x loading buffer is added. The SDS-PAGE was carried with a 12% precast polyacrylamide gel. Coomassie bright blue stain is used to observe the bands.
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#
  
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===IV Results===
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== Results ==
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'''sfGFP'''
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'''1. Confirmation of the Gene Circuit with sfGFP'''
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[[File:T--iBowu-China--2021lacI-2.png|thumb|center|600px|'''Figure.2. The expression of sfGFP with lacI+sfGFP can be controlled by iPTG induction.''' ]]
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Under the condition that there exists the part LacI, and there also exists LacO in the upstream gene of sfGFP, Iptg induction can control the switch on and off of the expression of green fluorescence protein sfGFP.
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[[File:T--iBowu-China--2021lacI-3.png|thumb|center|600px|'''Figure.3. The expression of sfGFP with lacI+sfGFP can be switched on with different concentrations of Iptg induction.''' ]]
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[[File:T--iBowu-China--2021bG-2.png|thumb|center|600px|'''Figure.2. Expression of GFP by the designed gene circuit with and without Iptg induction.''' ]]
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The plot and photo demonstrate that 0.5nM Iptg added into the medium is sufficient to induce the expression of sfGFP with the existence of lacI part.
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'''Enzyme activity of &beta;-glucuronidase'''
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First we needed to experimentally confirm that our gene circuit would work as we had expected. We used sfGFP (Part:K515105) gene to substitute the &beta;-glucuronidase gene in the above plasmid construction. The reconstructed plasmid was then cloned in DH5a and then extracted to be transformed into BL21(DE3). With the induction Iptg added, we can clearly measure strong signal of green fluorescence light. The intensity is about 100 fold stronger than the background, which is obtained by measuring the same bacteria culture but without Iptg induction. This result confirm that with Iptg induction, our gene circuit works normally and can be used for expression of bG enzyme.  
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[[File:T--iBowu-China--2021lacI-4.png|thumb|center|600px|'''Figure.4. The expression of &beta;-glucuronidase
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with lacI+sfGFP can be switched on with Iptg induction. The SDS-PAGE results clearly showed the expression of the enzynme at the indicated band. ''' ]]
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[[File:T--iBowu-China--2021lacI-5.png|thumb|center|600px|'''Figure.5. The expression of &beta;-glucuronidase induced by Iptg with the existence of LacI can be measured by the enzyme activity. Higher the total activity can be seen directly by deeper purple color. The activity can also be measured at OD540 with OD600 normalization of bacteria concentration.''' ]]
 
  
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In the scenario where there is LacI in the plasmid and also where there exists LacO in the upstream gene of &beta;-glucuronidase, this enzyme can be expressed in E. coli BL21 with Iptg induction. The enzymic activities and therefore the catalytic capabilities of the cells and the bacteria culture can therefore be controlled with different concentrations of induction substance.
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'''1. Successful Expression of the Protein with SDS-PAGE'''
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图：sds-page图
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随后，通过SDS-page，我们证实bG基因可以正常表达出蛋白。
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'''1. Confirmation of the Gene Circuit with sfGFP'''
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图：酶活图
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大肠杆菌的bG酶具有较好的酶活，而米曲霉和古生菌的bG酶有文献报道具有较好的酶活，但我们发现其在大肠杆菌中活性较低。
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<b>Summary</b>
 
<b>Summary</b>

Revision as of 04:56, 18 October 2021


6xHis-Beta-glucuronidase (E.coli, gusA)

6xHis-Beta-glucuronidase (E.coli, gusA)

Design

Figure.1. The plasmid constructions used for the expression of this part. This part encodes an enzyme β-glucuronidase.


Introduction

This year team iBowu-China used this part to express the enzyme called β-glucuronidase in E. coli strain BL21(DE3) and similar variants of the strain. The designed purpose of this enzyme is to convert glycyrrhizic acid into glycyrrhetinic acid, which is the effective active ingredient of licorice.

Protocol

  1. Transform the plasmids into E. coli BL21(DE3)
  2. Pick a single colony by a sterile tip from each of the LB plates for all the experimental and control groups. Add the colony into 4ml LB medium with kanamycin. Add 1mM iPTG to all experimental groups. Incubate at 37℃ in a shaker overnight.
  3. Add 100 µl bacteria culture medium into a sterile 96-well plate. Measure fluorescence at 488nm excitation and 502nm absorption, and then also OD600 for normalization, with a microplate reader.


  1. SDS-PAGE. The cells are lysed by 150 μL 4% SDS for 5 min at room temperature, then 10 min at 95℃. Then 6x loading buffer is added. The SDS-PAGE was carried with a 12% precast polyacrylamide gel. Coomassie bright blue stain is used to observe the bands.

Results

1. Confirmation of the Gene Circuit with sfGFP

Figure.2. Expression of GFP by the designed gene circuit with and without Iptg induction.

First we needed to experimentally confirm that our gene circuit would work as we had expected. We used sfGFP (Part:K515105) gene to substitute the β-glucuronidase gene in the above plasmid construction. The reconstructed plasmid was then cloned in DH5a and then extracted to be transformed into BL21(DE3). With the induction Iptg added, we can clearly measure strong signal of green fluorescence light. The intensity is about 100 fold stronger than the background, which is obtained by measuring the same bacteria culture but without Iptg induction. This result confirm that with Iptg induction, our gene circuit works normally and can be used for expression of bG enzyme.


1. Successful Expression of the Protein with SDS-PAGE 图：sds-page图 随后，通过SDS-page，我们证实bG基因可以正常表达出蛋白。


1. Confirmation of the Gene Circuit with sfGFP 图：酶活图 大肠杆菌的bG酶具有较好的酶活，而米曲霉和古生菌的bG酶有文献报道具有较好的酶活，但我们发现其在大肠杆菌中活性较低。


Summary LacI can effectively work together with LacO to control gene expression in E. coli BL21(DE3). Our results confirmed 0.5 to 2mM Iptg can be effective and sufficient to release the suppression of gene expression exerted by LacI protein.



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]