Difference between revisions of "Part:BBa K1722009"
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hTERT, which is short for human telomerase reverse transcriptase, is a cancer-cell specific promoter. It can be activated inside cancer cells with no effect on normal cells. By mutating four base pairs of hTERT sequence, we achieved an improved promoter with higher promote efficiency named super hTERT(shTERT). As an important component of human telomerase, hTERT express only in tumor cells and other immortal cells which are telomerase positive. Only in tumor cells that can express TERT can the promoter being activated to realize targeted expression of effector gene.
 
hTERT, which is short for human telomerase reverse transcriptase, is a cancer-cell specific promoter. It can be activated inside cancer cells with no effect on normal cells. By mutating four base pairs of hTERT sequence, we achieved an improved promoter with higher promote efficiency named super hTERT(shTERT). As an important component of human telomerase, hTERT express only in tumor cells and other immortal cells which are telomerase positive. Only in tumor cells that can express TERT can the promoter being activated to realize targeted expression of effector gene.
  
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In this plasmid, we construct hTERT with Green Fluorescent Protein(GFP), which is a widely used reporter gene. When shTERT promoter is activated, GFP is produced and be oxidized to fluoresce. By inserting this plasmid into T24 and 5637, two lines of bladder cancer cells, we are able to acquire GFP. Green fluorescent light is detected using confocal laser scanning microscopy. This indicates that hTERT promoter is able to be activated inside bladder cancer cells.
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In this plasmid, we construct hTERT with Green Fluorescent Protein(GFP), which is a widely used reporter gene. When shTERT promoter is activated, sfGFP is produced and be oxidized to fluoresce. By inserting this plasmid into T24 and 5637, two lines of bladder cancer cells, we are able to acquire sfGFP. Green fluorescent light is detected using confocal laser scanning microscopy. This indicates that shTERT promoter is able to be activated inside bladder cancer cells.
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shTERT is 454bp in length. <b>Fig. 1</b> shows the DNA sequence of shTERT is successfully amplified by PCR from psi-Check2 vector. From this electrophoretogram, we can see the brightness of shTERT PCR product is rather high compared with DNA Marker, which indicates that the PCR product of shTERT is in a high concerntration.
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<figure style="text-align: center"><img style="width:30%" src="https://static.igem.org/mediawiki/2015/1/18/%E9%AB%98%E6%95%88tert_pcr2.png"/><figcaption style="text-align:center"><b>Figure 1.</b> Electrophoretic analysis of PCR produution of hTERT promoter from psi-Check2. <figcaption style="text-align:center">(1:DL2000 DNA Marker 2:PCR production )</figcaption></figure>
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We then performed single digest(EcoRI) and double digest(EcoRI & PstI) to identify our pSB1C3-shTERT plasmid.<b>(Fig. 3)</b> From the eletrophoretogram, we have two electrophoresis strips at about 1172bp and 2070bp, which are exactly the length of shTERT+sfGFP and pSB1C3, respectively in Track 1 and a strip at about 3242bp in Track 2. From this enzyme cutting result, we could make sure the Gene sequence of shTERT+sfGFP succeeded in being constructed into pSB1C3 vector.
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<figure style="text-align: center"><img style="width:30%" src="https://static.igem.org/mediawiki/2015/1/1d/%E9%AB%98%E6%95%88tert%2Bgfp.png"/><figcaption style="text-align:center"><b>Figure 3.</b> Identification of recombinant plasmids pSB1C3-shTERT-sfGFP by one and two restriction enzymes. <figcaption style="text-align:center">[1:pSB1C3-shTERT-sfGFP double digest(EcoRI and PstI) 2:pSB1C3-shTERT-sfGFP single digest(EcoRI) 3:DL2000 DNA Marker]</figcaption></figure>
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<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Revision as of 06:41, 5 September 2015

shTERT+GFP Composite

hTERT, which is short for human telomerase reverse transcriptase, is a cancer-cell specific promoter. It can be activated inside cancer cells with no effect on normal cells. By mutating four base pairs of hTERT sequence, we achieved an improved promoter with higher promote efficiency named super hTERT(shTERT). As an important component of human telomerase, hTERT express only in tumor cells and other immortal cells which are telomerase positive. Only in tumor cells that can express TERT can the promoter being activated to realize targeted expression of effector gene.

In this plasmid, we construct hTERT with Green Fluorescent Protein(GFP), which is a widely used reporter gene. When shTERT promoter is activated, sfGFP is produced and be oxidized to fluoresce. By inserting this plasmid into T24 and 5637, two lines of bladder cancer cells, we are able to acquire sfGFP. Green fluorescent light is detected using confocal laser scanning microscopy. This indicates that shTERT promoter is able to be activated inside bladder cancer cells.

shTERT is 454bp in length. Fig. 1 shows the DNA sequence of shTERT is successfully amplified by PCR from psi-Check2 vector. From this electrophoretogram, we can see the brightness of shTERT PCR product is rather high compared with DNA Marker, which indicates that the PCR product of shTERT is in a high concerntration.

Figure 1. Electrophoretic analysis of PCR produution of hTERT promoter from psi-Check2.
(1:DL2000 DNA Marker 2:PCR production )

We then performed single digest(EcoRI) and double digest(EcoRI & PstI) to identify our pSB1C3-shTERT plasmid.(Fig. 3) From the eletrophoretogram, we have two electrophoresis strips at about 1172bp and 2070bp, which are exactly the length of shTERT+sfGFP and pSB1C3, respectively in Track 1 and a strip at about 3242bp in Track 2. From this enzyme cutting result, we could make sure the Gene sequence of shTERT+sfGFP succeeded in being constructed into pSB1C3 vector.

Figure 3. Identification of recombinant plasmids pSB1C3-shTERT-sfGFP by one and two restriction enzymes.
[1:pSB1C3-shTERT-sfGFP double digest(EcoRI and PstI) 2:pSB1C3-shTERT-sfGFP single digest(EcoRI) 3:DL2000 DNA Marker]

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 SapI.rc site found at 473

Design Notes

The shTERT promoter that is achieved from Shenzhen Sencond People's Hospital is constructed in psi-Check2 vector. To assemble it with GFP and pSB1C3, we have to design primers with certain restriction enzyme cutting site and amplify it. Then assemble the three parts using 3A Assembly Method.

Source

This composite part is assembled using shTERT and GFP. shTERT promoter is achieved from Shenzhen Second People's Hospital and GFP is achieved from 2015 Distribution Kit. We constructed the two DNA sequence in pSB1C3, the required vector.