Composite
UPll+AckRS

Part:BBa_K1722007

Designed by: Hui Ai   Group: iGEM15_SZU_China   (2015-09-02)

hUPll+AckRS Composite
Function Produce tRNA synthetase
Use in Bladder cells
RFC standard RFC 10
Backbone pSB1C3
Submitted by SZU_China 2015
hUPll+AckRS Composite

hUPll+AckRS, the device that we constructed this year, will perform its function with two other devices hTERT+tRNA(BBa_K1722010)/ shTERT+tRNA(BBa_K1722011) and SV40+Rlu(BBa_K1722012).

hUPll is a bladder tissue-specific promoter being found in human urothelium. Uroplakin II (UPII) has been characterized as a bladder tissue-specific protein[1] and the expression of uroplakin II was found to be limited to bladder-derived cells.[2-3] Other members of uroplakins, including uroplakinla(UPla), uroplakinlb(UPlb), and uroplakinlll(UPlll), have also been characterized. Therefore, the promoters that direct the expression of the uroplakins may be useful in constructing tissue-specific vectors for bladder cancer gene therapy. Research shows that most of thecis elements that confer the bladder-specificity and differentiation-dependent expression of the human UPll gene reside in the 2542-bp sequence, and TNF driven by the human UPll(hUPll) promoter is effective in the specific inhibition of bladder cancer growth both in vivo and in vitro. Zhu et al transtected the plasmid phUPll-EGFP containing DNA fragment(hUPll) into bladder cancer(BIU-87), renal carcinoma(GRC-1) and endothelial(EC) cell lines.[4](Fig. 1)

Figure 1. The plasmid phUPII-EGFP containing DNA fragment (hUPII) 2542 bp upstream of the UPII gene was transfected into bladder cancer (BIU-87), renal carcinoma (GRC-1), and endothelial (EC) cell lines. Transient transfection was determined either by confocal microscopy or flow cytometric analysis of GFP expression. (a)The activity of human UPII promoter in BIU-87, GRC-1, and EC cell lines. Positive green signals from UPII were more and stronger in BIU-87 than in EC and GRC-1 cell lines.(b) The GFP activity in BIU-87, GRC-1, and EC was tested by flow cytometry. A positive rate from UPII was higher in BIU-87 than in EC and GRC-1 cell lines. The percentage of GFP-positive cells in BIU-87 cell line, EC cell line, and GRC-1 cell line was 10.1, 1.8, and 0% respectively.[4]

Ack is a kind of unnatural amino acid(UAA) that is close structural analog of Lysine, a canonocal amino acid. In the orthogonal system of the project of 2015 SZU-iGEM, the construction of our UAA orthogonal system rely on an orthogonal pair of tRNA(CUA) and an AckRS charging the tRNA with Ack. tRNA(CUA) has an anticodon CUA, which can pair with UAG, the amber mutated stop codon, perfectly. With the help of AckRS, the unnatural amino acid Ack can be incorporated into proteins. aaRS functioning in the form of polycomplex in living cells.[5-6] Research on Structural Biology and Bioinformatics shows that aaRS can combine with other proteins, forming highly organized complex, to be involved in many vital physiological processes.[5-6] In the last decade, methods for the translational incorporation of UAAs using orthogonal aaRS-tRNA(CUA) pairs were developed. With modified aaRS which can specifically recognise a type of UAA, the UAA can be site-specifically incorporated to produce a protein with new structure and function or to expand the genetic code. The wild-type pyrrolysyl-tRNA synthatase(PylRS) from Methanosarcina mazei readily accepts a number of lysine derivatives as substrates. This enzyme can further be engineered by mutagenesis to utilize a range of UAAs[7] and the AckRS that we used in our project is one of them.

We constructed hUPll and AckRS in this plasmid to initiate the expression of AckRS inside bladder cells. When both the hUPll and hTERT promoters are activated in bladder cancer cells, the whole orthogonal system that we have constructed can work efficiently. Our unnatural amino acid orthogonal system consists of three devices(plasmids).

(1)hUPll+AckRS(BBa_K1722007): hUPll is a bladder-cell specific promoter. when it's activated, AckRS, a tRNA synthetase, will be produced.

(2)hTERT+tRNA(BBa_K1722010)/ shTERT+tRNA(BBa_K1722011): hTERT and shTERT are cancer-cell specific promoters. tRNA can be expressed out when the promoter is activated.

(3)SV40+Rluc(BBa_K1722012): SV40 is a widely used strong promoter. Rluc is a reporter that can produce RLUC which is a kind of luciferase.

There is an amber stop codon UAG in the sequence of Rluc. The tRNA that is produced from the second plasmid has CUA as its anticodon, which can pair with the stop codon of the mRNA chain of Rluc. AckRS can achieve the attachment of Ack, the unnatural amino acid, and the tRNA. In this way, when all the three promoters are activated inside bladder cancer cell, Ack can be charged to the specific tRNA and then the anticodon of tRNA can pair with the stop codon on the mRNA chain of Rluc. In natural condition, this Rluc gene cannot be fully expressed because of the amber stop codon. However, with our orthogonal system, it can be produced and detected.

The three plasmids that we've constructed were inserted into three lines of cells(HFC, Hela and 5637). HFC is short for Human Fiber Cells, which is a kind of normal cell in human bladder. Hela and 5637 are cervical cancer cell line and bladder cancer cell line, respectively. We set two groups in each cell lines, one have Ack, an unnatural amino acid, in the culture medium, and another do not have it. The luminescence intensity of 5637 with Ack in the medium is much higher than other groups, which indicates that the specificity of the two promoters are high enough and our orthogonal system is working.(Fig. 2)

Figure 2. Renilla Luminescence

We also constructed two plasmids and three plasmids system with GFP as their reporter gene. These systems were transfected into T24, a kind of bladder cancer cell. The working efficiency of our orthogonal system can be detected by comparing the luminescence intensity of green fluorescent of each groups. Two plasmids system is composed of hUPll-AckRS-GFP(amber mutated) and hTERT-tRNA. And the construction members of three plasmids system include hUPll-AckRS, hTERT-tRNA and SV40-GFP(amber mutated). Each system is divided into two groups: one have Ack in the medium and another one do not have Ack. Adding a positive control group, which is constructed by hUPll-GFP(wild-type), we have five groups of bladder cancer cells in total. Luminescent intensity results are shown in Fig. 3.

Figure 3. Green fluorescent intensity.
(A: Positive Control Group B: Two Plasmids-no Ack Group C: Two Plasmids-with Ack Group D: Three Plasmids-no Ack Group E: Three Plasmids-with Ack Group

As we can see, no cell in Two Plasmids-no Ack Group and Three Plasmids-no Ack Group produce green fluorescent light. In Two Plasmids-with Ack Group and Three Plasmids-with Ack Group, however, is full of luminescent cells. From this result, we can tell our orthogonal system work efficiently.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 1622
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 811
  • 1000
    COMPATIBLE WITH RFC[1000]

Design Notes

The AckRS gene that is achieved from Shenzhen Second People's Hospital has two EcoR1 restriction enzyme cutting sites in its sequence. We mutated them and designed primers to amplified both hUPll and AckRS from psi-Check2 vector. Then 3A Assembly was used to construct these two gene in pSB1C3.

Source

We achieved both hUPll promoter and AckRS from Shenzhen Second People's Hospital.

References

[1]Wu XR, Lin JH, Walz T, et al. Mammalian uroplakins, a group of highly conserved urothelial differentiation related membrane proteins. J Biol Chem. 1994;269:13716–13724.

[2]Yuasa T, Yoshiki T, Isono T, et al. Expression of transitional cell specific genes uroplakin Ia and II in bladder cancer detection of circulating cancer cells in the peripheral blood of metastatic patients. Int J Urol. 1999;6:286–292.

[3]Moll R, Wu XR, Lin JH, Sun TT. Uroplakins specific membrane proteins of urothelial umbrella cells as histological markers of metastatic transitional cell carcinomas. Am J Pathol. 1995;147:1383–1397.

[4]Zhu H J, Zhang ZQ, Zeng XF, et al. Cloning and analysis of human uroplakin II promoter and its ap plication for gene therapy in bladder cancer[J] Cancer Gene Ther, 2004, 11: 263-272

[5] Hausmann C D, Ibba M. Aminoacyl- tRNA synthetase complexes: molecular multitasking revealed. FEMS Microbiol Rev, 2008, 32(4): 705-721

[6] Han JM, Kim J Y, Kim S. Molecular network and functional implications of macromolecular tRNA synthetase complex. Biochem Biophys Res Commun, 2003, 303(4): 985-993

[7] Veronika F, Milan V, Sabine S. Structural basis for the site-specific Incorporation of Lysine Derivatives into Proteins. Plos One, 2014, 9(4): 1-7


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