Difference between revisions of "Part:BBa I13522"
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|Can synthesize over 200pmols of GFPmut3b in 6 hours
 
|Can synthesize over 200pmols of GFPmut3b in 6 hours
 
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|}
 +
 +
='''IISER Kolkata 2019'''=
 +
 +
=== The Characterization of BBa_K381001 ===
 +
 +
<p>
 +
    In our project to combat Leishmaniasis using a genetically modified
 +
    bacterium we planned to use bacterial promoter pTET as a upstream of our
 +
    coding region but for it to act effectively we need to quantify that its
 +
    promoter activity is comparable in both bacterial and mammalian culture
 +
    medium.
 +
</p>
 +
<p>
 +
    <strong>Aim</strong>
 +
</p>
 +
<p>
 +
    BBa_I13522 was used as a test circuit to compare the activity of pTET
 +
    bacterial promoter in bacterial medium Luria Broth and mammalian cell
 +
    culture medium DMEM.
 +
</p>
 +
<p>
 +
    <strong>Method</strong>
 +
</p>
 +
<p>
 +
    E. coli DH5α were transformed using re-suspended BBa_I13522 pSB1C3 plasmids
 +
    from iGEM 2019 distribution kit and selected using Chloramphenicol LB agar
 +
    plates.
 +
</p>
 +
<ol>
 +
    <li>
 +
            Single positive colony of BBa_I13522 was added to 10ml of LB media
 +
            and left overnight at 37° C on 150 rpm in an incubator. Along with
 +
            this, a positive control with non-transformed E coli cells was also
 +
            kept in liquid culture in the similar conditions.
 +
        </li>
 +
    <li>
 +
            100 µL of overnight culture was added to 10ml of LB media and DMEM
 +
            media in two different test tubes. For positive control secondary
 +
            culture was added in 10ml of LB media and all the test tubes were
 +
            left in incubator for 4 hours until the OD for the control reached
 +
            0.6
 +
        </li>
 +
    <li>
 +
            2 mL of liquid culture from each of the test tube was centrifuged,
 +
            washed and re-suspended in 500 µL of Phosphate-Buffered Saline
 +
            (PBS)
 +
        </li>
 +
    <li>
 +
            100uL of the re-suspended culture was added to each well of 96 well
 +
            plate.
 +
        </li>
 +
    <li>
 +
            Varioskan LUX multimode reader was used to measure the GFP
 +
            fluorescence: Excitation at 494 nm and emission at 525 nm.
 +
        </li>
 +
</ol>
 +
<p>
 +
    <strong>Result</strong>
 +
</p>
 +
<p>
 +
    BBa_I13522 transformed <em>E. coli</em> shows similar GFP expression in
 +
    both bacterial and mammalian media.
 +
</p>
 +
<figure>
 +
<img class="bigimage" src="https://2019.igem.org/wiki/images/5/53/T--IISER_Kolkata--ptet_gfp.JPG"/>
 +
<figcaption>Mean GFP fluorescence (Excitation 494nm/Emission 525nm) of BBa_I13522 transformed E.coli grown in two different media as compared untransformed E. coli grown in LB media and suspended in PBS</figcaption>
 +
</figure>
 +
<p>
 +
    <strong>Conclusion</strong>
 +
</p>
 +
<p>
 +
    The bacterial promoter pTET constitutively expresses comparable amount the
 +
    downstream coding region (GFP in this case) irrespective of medium in which
 +
    it is grown.
 +
</p>

Revision as of 10:39, 20 October 2019

pTet GFP

Untagged GFP behind a constitutive promoter.

Usage and Biology

GFP only control

The version shipped with the 2008 iGEM distribution does not appear to function. Unclear why. Jkm 18:40, 22 August 2008 (UTC)

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
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 724


Pictures

  • BBa_I13522 visualized under non-UV lightbox

  • BBa_I13522 visualized under 254nm wavelength UV lightbox

Characterization of Cell-Free Chassis

This part was used to characterize the cell-free chassis. Details of characterization can be found on the cell-free characterization page. Other interesting isothermal properties of this construct are available at the [http://2007.igem.org/Imperial/Cell_by_Date/Testing Cell By Date project page] of Imperial 2007 wiki.

Parameter Value and Description
Optimal Temperature Expression increases with temperature from 4°C to 30°C
Optimal DNA Concentration Works optimally with 2µg of DNA in 60 µl of cell-extract
Peak Expression Time Peak rate of output occurs after approximately 85 minutes
Expression Lifespan Expression levels off after 2 hours
Expression Capacity Can synthesize over 200pmols of GFPmut3b in 6 hours

IISER Kolkata 2019

The Characterization of BBa_K381001

In our project to combat Leishmaniasis using a genetically modified bacterium we planned to use bacterial promoter pTET as a upstream of our coding region but for it to act effectively we need to quantify that its promoter activity is comparable in both bacterial and mammalian culture medium.

Aim

BBa_I13522 was used as a test circuit to compare the activity of pTET bacterial promoter in bacterial medium Luria Broth and mammalian cell culture medium DMEM.

Method

E. coli DH5α were transformed using re-suspended BBa_I13522 pSB1C3 plasmids from iGEM 2019 distribution kit and selected using Chloramphenicol LB agar plates.

  1. Single positive colony of BBa_I13522 was added to 10ml of LB media and left overnight at 37° C on 150 rpm in an incubator. Along with this, a positive control with non-transformed E coli cells was also kept in liquid culture in the similar conditions.
  2. 100 µL of overnight culture was added to 10ml of LB media and DMEM media in two different test tubes. For positive control secondary culture was added in 10ml of LB media and all the test tubes were left in incubator for 4 hours until the OD for the control reached 0.6
  3. 2 mL of liquid culture from each of the test tube was centrifuged, washed and re-suspended in 500 µL of Phosphate-Buffered Saline (PBS)
  4. 100uL of the re-suspended culture was added to each well of 96 well plate.
  5. Varioskan LUX multimode reader was used to measure the GFP fluorescence: Excitation at 494 nm and emission at 525 nm.

Result

BBa_I13522 transformed E. coli shows similar GFP expression in both bacterial and mammalian media.

<figure> <img class="bigimage" src="T--IISER_Kolkata--ptet_gfp.JPG"/> <figcaption>Mean GFP fluorescence (Excitation 494nm/Emission 525nm) of BBa_I13522 transformed E.coli grown in two different media as compared untransformed E. coli grown in LB media and suspended in PBS</figcaption> </figure>

Conclusion

The bacterial promoter pTET constitutively expresses comparable amount the downstream coding region (GFP in this case) irrespective of medium in which it is grown.