Difference between revisions of "Part:BBa K3560002"

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<p><b>1 Plasmid construction </b></p>
 
<p><b>1 Plasmid construction </b></p>
 
We used PRADK-PgroES as a vector (6010bp). Using recombinant plasmid PCDF-1-eGFP as template，and eGFP-F and eGFP-R as primers, eGFP fragment (717bp) was obtained by amplification. Gibson assembly was used to ligate eGFP fragment with the PRADK-PgroES linearized vector. Colony PCR (775bp) was used for the transformed colonies using PgroES-eGFP-PF and PgroES-eGFP-PR as primers. The positive colonies were transferred, and the plasmids were extracted. Gene sequencing was used to verify the plasmid PRADK-PgroES-eGFP.
 
We used PRADK-PgroES as a vector (6010bp). Using recombinant plasmid PCDF-1-eGFP as template，and eGFP-F and eGFP-R as primers, eGFP fragment (717bp) was obtained by amplification. Gibson assembly was used to ligate eGFP fragment with the PRADK-PgroES linearized vector. Colony PCR (775bp) was used for the transformed colonies using PgroES-eGFP-PF and PgroES-eGFP-PR as primers. The positive colonies were transferred, and the plasmids were extracted. Gene sequencing was used to verify the plasmid PRADK-PgroES-eGFP.
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Table 1 Primer sequences
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<p><b>2 Experimental results </b></p>
 
<p><b>2 Experimental results </b></p>
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The transfected D. radiodurans (notated as PgroES in Figure 3) were cultured till OD600 reached 0.5 and 1.0, and then were taken for fluorescence intensity detection. D. radiodurans strain with empty plasmid PRADK was as a blank control (notated as Control). The fluorescence intensity of PgroES under OD600=0.5 and OD600=1.0 was 55745和67807.3. PgroES showed stronger intensity than Control.
 
The transfected D. radiodurans (notated as PgroES in Figure 3) were cultured till OD600 reached 0.5 and 1.0, and then were taken for fluorescence intensity detection. D. radiodurans strain with empty plasmid PRADK was as a blank control (notated as Control). The fluorescence intensity of PgroES under OD600=0.5 and OD600=1.0 was 55745和67807.3. PgroES showed stronger intensity than Control.
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  <div class="unterschrift"><b>Figure 2 Fluorescence microscopic image of D. radiodurans transfected with PRADK-PgroES-eGFP recombinant plasmid</b>
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  <div class="unterschrift"><b>Figure 3 Fluorescence intensity of D. radiodurans transfected with PRADK-PgroES-eGFP recombinant plasmid (PgroES) and D. radiodurans transfected with empty plasmid PRADK (Control)</b>
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<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  
 
===Functional Parameters===
 
===Functional Parameters===

Latest revision as of 07:17, 10 October 2023


PGroES

PGroES is a promoter which can be recognized in Deinococcus radiodurans, which expresses the downstream gene.

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
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 190
  • 1000
    COMPATIBLE WITH RFC[1000]

Contribution

Group: 2023iGEM Nanjing-SDG

Author: Kewei Li

Summary: Characterization of the strength of PgroES promoter in driving the expression of eGFP by fluorescence intensity detection of the recombinant plasmid

Characterization

We used PRADK-PgroES as the vector and eGFP fragment amplified from the template of PCDF-1-eGFP, to construct recombinant plasmid PRADK-PgroES-eGFP. This plasmid was used to express eGFP in Deinococcus radiodurans (D. radiodurans). The fluorescence intensity of the transfected strain was analyzed to characterize the expression of eGFP with PgroES promoter.

Experiments Results

1 Plasmid construction

We used PRADK-PgroES as a vector (6010bp). Using recombinant plasmid PCDF-1-eGFP as template，and eGFP-F and eGFP-R as primers, eGFP fragment (717bp) was obtained by amplification. Gibson assembly was used to ligate eGFP fragment with the PRADK-PgroES linearized vector. Colony PCR (775bp) was used for the transformed colonies using PgroES-eGFP-PF and PgroES-eGFP-PR as primers. The positive colonies were transferred, and the plasmids were extracted. Gene sequencing was used to verify the plasmid PRADK-PgroES-eGFP.

Table 1 Primer sequences

Figure 1 Construction of PRADK-PgroES-eGFP recombinant plasmid

2 Experimental results

PgroES is a common promoter in Deinococcus radiodurans (D. radiodurans). eGFP encodes enhanced green fluorescent protein which is used as a reporter protein since it emits green fluorescence when exposed to light in the blue to ultraviolet range. PRADK-PgroES-eGFP recombinant plasmid is used to express eGFP using PgroES as the promoter.

PRADK-PgroES-eGFP was transfected into D. radiodurans, notated as PgroES in figures. The transfected D. radiodurans were cultured in TGY medium for 48h. The fermentation broth was centrifuged, washed, and resuspended in PBS, and then the diluted bacterial solution was smeared on a glass slide. By using a fluorescence microscope, green fluorescence was observed (Figure 2).

The transfected D. radiodurans (notated as PgroES in Figure 3) were cultured till OD600 reached 0.5 and 1.0, and then were taken for fluorescence intensity detection. D. radiodurans strain with empty plasmid PRADK was as a blank control (notated as Control). The fluorescence intensity of PgroES under OD600=0.5 and OD600=1.0 was 55745和67807.3. PgroES showed stronger intensity than Control.

Figure 2 Fluorescence microscopic image of D. radiodurans transfected with PRADK-PgroES-eGFP recombinant plasmid

Figure 3 Fluorescence intensity of D. radiodurans transfected with PRADK-PgroES-eGFP recombinant plasmid (PgroES) and D. radiodurans transfected with empty plasmid PRADK (Control)

This is a new basic part, which expresses the downstream gene.

The results are shown in BBa_K3560004 and BBa_K3560005.