First, we designed a sensitive system called hepashield to kill liver cancer cells.

To assay every part, we plan to use MTT method to test the relative activity of cells[1].

To check the activation of three promoters CMV, AFP, hTERT, we transfected plasmid pGL4.22-AFP, pGL4.22-hTERT and pGL4.22-CMV into three cell lines (HepG2, Huh7 and Lo2). Through experiments, we conclude that promoters AFP and hTERT are active Huh7 and HepG2, for they belong to HCC, but do not work in human normal hepatocellular, and less effective than CMV. Therefore, we did the cotransfection between pGL4.22-hTERT-GAL4-VP16 and pGL4.35 in these three cell lines.

AFP is invalid in Huh7 cell line(p>0.05), while another two promoters can drive luciferase fluoresces binding with zymolyte

After the preliminary try on promoters, we add GAL4-VP16 into the plasmid. We found that, these three promoters showed the possibility to make our system effective.
According to what we discussed above, we can draw some conclusions here.
The good news is that CMV promoter and GAL4-VP16 system really have positive effect on those three cell lines while the negative part is that the specificity of our double-switch system with AFP and hTERT is not as high as we expected.
But where does the problem come from? We come up with several proposals:
1. Promoter gene code. The Transfection efficiency is not good enough because of unsatisfying cell condition[2]. The culture of these cells is also a big challenge for us. We cloned the code of AFP and hTERT from ATG, and maybe with the Upstream noncoding region can make sense.
2. GAL80. Though we are pretty confident in regarding the GAL80 as a good inhibitor to GAL4 protein, but the result is far away from our expectation. We suppose that there are some barriers in the process of expression of GAL80[3]. However, we will try to explore it later!
Although we have repeated some steps of our experiments with several times of trial and error, the deficiency of time does not allow us to improve the system adequately.
Apart from what we mentioned above, we also plan to do the following tests:
1. Clone AFP and hTERT code with extra 200bp of noncoding region before ATG.
2. Adjust the transfer condition. For example, maybe in the condition of high cell density, cells will secrete more cytokines, and then hepatocellular carcinoma cells can promote the growth of the cells through paracrine pathways.
3. Use Tetracycline expression system to substitute GAL80[4].
4. Do experiment on the remaining part: GAL4 Binding domain (BD) and herpes simlex virus-thymidine kinase (HSV-TK)

HepG2 is an immortalized cell line consisting of human liver carcinoma cells, derived from the liver tissue of a 15-year-old Caucasian male who had a well-differentiated hepatocellular carcinoma. The morphology of HepG2 cells is epithelial and contains 55 chromosome pairs. HepG2 cells can be raised successfully at a large scale, and secrete many plasma proteins, such as transferrin, fibrinogen, plasminogen and albumin. They can be stimulated with human growth hormone. HepG2 cells are adherent, epithelial-like cells growing as monolayers and in small aggregates[5].

HuH-7 is a well differentiated hepatocyte derived cellular carcinoma cell line that was originally taken from a liver tumor in a 57-year-old Japanese male in 1982. HuH-7 cell is HBV negative，and can produce some cytoplasmic proteins, such as albumin, a antitrypsin, AFP, etc[6].

Once we felt that we could understand and control GAL4-VP16, an unusually potent transcriptional activator, we would like to make our system more accessible to future iGEM teams. Although the part we submitted is inherently easy to clone and implement, as it with strong promoter CMV and GAL4-VP16, we would like to make it even more easier to implement. With this thought, we created a construction Bba_K2580666 (CMV-GAL4-VP16) and added them to the registry. This ready-to-clone parts should make it cheaper and easier for future teams to make an innovative synthetic biological tool.

[1] Sladowski D, Steer S J, Clothier R H, et al. An improved MTT assay[J]. J.immunol.methods, 1993, 157(1-2):203.
[2]Jiang M, Chen G. High Ca2+-phosphate transfection efficiency in low-density neuronal cultures[J]. Nature Protocols, 2006, 1(2):695.
[3]Leuther K K, Johnston S A. Nondissociation of GAL4 and GAL80 in vivo after galactose induction.[J]. Science, 1992, 256(5061):1333-1335.
[4]Frost H M. Tetracycline-based histological analysis of bone remodeling.[J]. Calcif Tissue Res, 1969, 3(1):211-237.
[5]Cederbaum A I, Wu D, Mari M, et al. CYP2E1-dependent toxicity and oxidative stress in HepG2 cells  ;  , 1, 2[J]. Free Radic Biol Med, 2001, 31(12):1539-1543.
[6]Shih C M, Lo S J, Miyamura T, et al. Suppression of hepatitis B virus expression and replication by hepatitis C virus core protein in HuH-7 cells.[J]. Journal of Virology, 1993, 67(10):5823-5832.