Difference between revisions of "Part:BBa K3332085"

 
 
(15 intermediate revisions by 3 users not shown)
Line 3: Line 3:
 
<partinfo>BBa_K3332085 short</partinfo>
 
<partinfo>BBa_K3332085 short</partinfo>
  
−
A composite part to check the function of PLtetO promoter.
+
A composite part to check the function of pLtetO-1 promoter.
 +
 
 +
With this part, the pLtetO-1 promoter can be tested by observing the fluorescence intensity.
  
−
<!-- Add more about the biology of this part here
 
 
===Usage and Biology===
 
===Usage and Biology===
 +
<html>
 +
    <figure>
 +
        <img src="https://2020.igem.org/wiki/images/b/bf/T--XMU-China--XMU-China_2020-gai.png" width="60%" style="float:center">
 +
        <figcaption>
 +
        <p style="font-size:1rem">
 +
        </p>
 +
        </figcaption>
 +
    </figure>
 +
</html>
 +
'''Fig 1.''' J23106_B0031_tetR_B0015_pLtetO-1_B0034_ecfp_B0015
 +
 +
This part can be used to test that if the pLtetO-1 promoter can work.
 +
 +
===Characterization===
 +
The agarose gel electrophoresis images of target fragments are shown as below：
 +
[[File:Fig.2 pLtetO-1_E0420_pSB1C3 and pUC57(BBa K3332084) digested by EcoR I and Pst I.png|none|500px|caption]]
 +
'''Fig 2.''' pLtetO-1_E0420_pSB1C3 and pUC57(BBa_K3332084) digested by ''Eco''R I and ''Pst'' I (about 1026 bp)
 +
[[File:2034 fig.3.png|none|500px|caption]]
 +
'''Fig 3.''' J23106_P0140_ pLtetO-1_E0420_pSB1C3(BBa_K3332085) digested by ''Spe'' I and ''Pst'' I (about 3922 bp)
 +
 +
'''Note：'''E0420 is equal to B0034_E0020_B0015
 +
 +
'''Protocol:'''
 +
 +
1. Preparation of stock solution
 +
 +
Dissolve ATc in absolute alcohol to make 1000× stock solution（the work concentration is 100 ng/mL）
 +
 +
2.Culture glycerol bacteria containing the corresponding plasmid in test tube for 12 h.
 +
 +
3.Add 4 mL of the above bacterial solution into 200 mL LB medium and maintain the culture condition at 37 ℃ and 180 rpm.
 +
 +
4.Add 200 µL ATc stock solution into the induction group when OD increased to 0.6.
 +
 +
5.Induce for 6 hours and the condition is the same as before.
 +
 +
6.Then, sampling 0.5 mL culture in each tube. All samples are centrifuged at 12000 rpm, 1 minute. Remove supernatant and add 500 µL sterile PBS to resuspend.
 +
 +
7.Measure the fluorescence intensity（ECFP）and corresponding OD<sub>600</sub> by 96-well plate reader, then calculate the fluorescence / OD value of each group.
 +
 +
 +
Here is the result:
 +
 +
<html>
 +
    <figure>
 +
        <img src="https://2020.igem.org/wiki/images/d/d5/T--XMU-China--XMU-China_2020-pLtetO.png" width="60%" style="float:center">
 +
        <figcaption>
 +
        <p style="font-size:1rem">
 +
        </p>
 +
        </figcaption>
 +
    </figure>
 +
</html>
 +
 +
'''Fig 4.''' Fluorescence intensity/OD<sub>600</sub> for induction and non-induction group (6 hours). Data are collected and analyzed according to iGEM standard data analysis form after 6 hours of induction.
 +
 +
In the figure, there is no obvious difference in fluorescence intensity/OD600 between induction group and non-induction group of the negative control (J23100) and pLtetO-1_E0420. While the fluorescence intensity/OD600 of J23106_P0140_pLtetO-1_E0420 in induction group is higher than non-induction group obviously. That is to say, 100ng/mL ATc can inhibit the repression of tetR on pLtetO-1, then turn on the expression of downstream gene of pLtetO-1.
 +
 +
===Reference===
 +
[1] Chan CT, Lee JW, Cameron DE, Bashor CJ, Collins JJ. 'Deadman' and 'Passcode' microbial kill switches for bacterial containment. Nat Chem Biol. 2016;12(2):82-86. doi:10.1038/nchembio.1979
  
 
<!-- -->
 
<!-- -->

Latest revision as of 22:45, 27 October 2020


J23106-RBS-tetR-pLtetO-1-ECFP-terminator

A composite part to check the function of pLtetO-1 promoter.

With this part, the pLtetO-1 promoter can be tested by observing the fluorescence intensity.

Usage and Biology

Fig 1. J23106_B0031_tetR_B0015_pLtetO-1_B0034_ecfp_B0015

This part can be used to test that if the pLtetO-1 promoter can work.

Characterization

The agarose gel electrophoresis images of target fragments are shown as below：

caption

Fig 2. pLtetO-1_E0420_pSB1C3 and pUC57(BBa_K3332084) digested by EcoR I and Pst I (about 1026 bp)

caption

Fig 3. J23106_P0140_ pLtetO-1_E0420_pSB1C3(BBa_K3332085) digested by Spe I and Pst I (about 3922 bp)

Note：E0420 is equal to B0034_E0020_B0015

Protocol:

1. Preparation of stock solution

Dissolve ATc in absolute alcohol to make 1000× stock solution（the work concentration is 100 ng/mL）

2.Culture glycerol bacteria containing the corresponding plasmid in test tube for 12 h.

3.Add 4 mL of the above bacterial solution into 200 mL LB medium and maintain the culture condition at 37 ℃ and 180 rpm.

4.Add 200 µL ATc stock solution into the induction group when OD increased to 0.6.

5.Induce for 6 hours and the condition is the same as before.

6.Then, sampling 0.5 mL culture in each tube. All samples are centrifuged at 12000 rpm, 1 minute. Remove supernatant and add 500 µL sterile PBS to resuspend.

7.Measure the fluorescence intensity（ECFP）and corresponding OD600 by 96-well plate reader, then calculate the fluorescence / OD value of each group.


Here is the result:

Fig 4. Fluorescence intensity/OD600 for induction and non-induction group (6 hours). Data are collected and analyzed according to iGEM standard data analysis form after 6 hours of induction.

In the figure, there is no obvious difference in fluorescence intensity/OD600 between induction group and non-induction group of the negative control (J23100) and pLtetO-1_E0420. While the fluorescence intensity/OD600 of J23106_P0140_pLtetO-1_E0420 in induction group is higher than non-induction group obviously. That is to say, 100ng/mL ATc can inhibit the repression of tetR on pLtetO-1, then turn on the expression of downstream gene of pLtetO-1.

Reference

[1] Chan CT, Lee JW, Cameron DE, Bashor CJ, Collins JJ. 'Deadman' and 'Passcode' microbial kill switches for bacterial containment. Nat Chem Biol. 2016;12(2):82-86. doi:10.1038/nchembio.1979

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
    COMPATIBLE WITH RFC[1000]