Difference between revisions of "Part:BBa K1456004"

Latest revision as of 03:06, 16 October 2018

Hypoxia Response Element(HRE)

• Hypoxia response elements (HREs) are 234 bp long small DNA sequences present in most of the body cells which work as binding domains for hypoxia inducible factor-1α (HIF-1α), a common transcription factor found in our body cells released during hypoxic conditions. (Semenza et al. 1992)
• We intend to add several HREs to the upstream region of weak constitutive promoter, PminiCMV, in order to design a novel hypoxia inducible promoter. We expect that in normal conditions with enough oxygen presence, PminiCMV promoter will express very small amount of mRNA which will be degraded soon conceivably. However, in hypoxic conditions, more transcription factors will hopefully bind to our designed HRE sites to enhance the transcription rate of PminiCMV promoter.
• According to the studies in the literature, HRE sequence has provided significant high transcription rate in hypoxic cells when it was attached on the upstream region of Simian virus 40 (SV40) promoter. (Tang et al. 2002) In this study, plasmids which contain HRE inducible promoters were presented in cell lines and the amount of these plasmids were increased gradually to test whether the detection level and transcription rate of hypoxia would raise. As a result, the production rate of reporter gene attached to HRE promoters increased 410 times more. This result encouraged us to use these sequences as novel, efficient and powerful hypoxic promoters in human cells.

Modeling

• In our promoter design, we added hypoxia response element to the upstream of our effector protein gene. In hypoxic conditions, HIF1-α protein levels are high enough to translocate it into the nucleus and form a transcriptional complex with the HIF1-β. We decided not to include a separate step for the dimerization and binding processes to hypoxia response element (HRE). We assumed the HIF1-α level as a constant in hypoxia which does not change dynamically. HIF1-α/ HIF1-β complex can bind to HRE which then activates the transcription of our gene interest. These are tPA, SOD, GPX, aprotinin or luciferase. In addition, CMV promoter has a leaky luciferase expression without HIF1-α. Therefore, we assumed that luciferase expression from HRE + CMV + Luciferase without HIF1-α is equal to the expression of CMV + Luciferase in the normoxic condition.
• These are the four chemical reactions which represent each process.

The symbol declaration is:

• X1 : HIF1-α
• Y : HRE- pCMV
• X1Y : HIF1-α + [HRE- pCMV] Complex
• X2 : Interested protein (in model, luciferase)
• X3 : Only pCMV Promoter (distincted from HRE)

Finally, we assigned each process with a reaction rate constant.

How did we build it?

• Using the SimBiology toolbox for Matlab, we designed a framework of the hypoxia inducible promoter system (Figure 1). The processes of binding, dissociation of transcription factor, expression of luciferase in hypoxic/normoxic conditions, and the degradation of luciferase were included in framework.

• After designing the basic framework of our model, we were required to establish mathematical equations for each reaction withan appropriate rate constants. These equations and the corresponding values are shown below in Tables 1 and 2.

What did it show?

• Before running the model, we had to decide on how the hypoxia response element affects gene expression The pCMV promoter (distincted from HRE) expressed approximately 0.15 nM and the [HRE- pCMV] complex expressed 1.86 nM of luciferase in 12 hours.In our simulations, we show that adding hypoxia response element in our promoter design increases the protein expression approximately 20 times more in hypoxic conditions.

• This data was then used to design an experiment that could be performed in the lab to verify the model. You can visit our results page to see the comparison.
• If we are to compare our modeling results (which appoints that the activity of luciferase will increase in hypoxic condition according to the parameters collected from literature research) with our experimental results, we can say that they are rising in a close manner of proportion. According to our modelling results, the minimal increase in our wet lab can be for the following reasons: Cell cultures exposed to hypoxic conditions in eukaryotic cells die because of failing to produce luciferase. The reversable mechanisms (feed back regulation mechanisms) which form the cell-life balance cannot function in hypoxic conditions therefore ,decreasing the speed rate of dependent enzymes and transcription factors. As an example, hıf1alph involved in the regulation of the operation of prolyl hydroxilase can be shown which changes according to the level of oxygen.[1]

Experimental Results

According to the results, in hypoxic conditions comparing with normoxia, HRE been inserted into pTRE-luciferase vector shows 7 times more activity by producing more luciferase in HepG2 cell line. In HEK293 cell line, this activity is measured in hypoxia 1,5 times more than normoxia. This results prove that HRE sequence improves the production rate of desired protein in hypoxia comparing with normal oxygen levels.

Improved Results

• Group: LZU-CHINA 2018
• Author: Xin Nie; Tuoyu Zhou
• Summary: We modified a new HRE opereon with 5 reapts following miniCMV promoter(BBa_K2796052). We also had detected the function of opreon HRE by monitoring the relative fluorescence intensity of copGFP with different CoCl(II) concentration.

• According to the experimental results(Figure 1 and 2), HRE promoter can induce higher copGFP fluorescence level under hypoxia conditions.But at high concentrations of CoCl(II), cells apoptosis as a result of metal toxicity, leading to a sharp decrease in fluorescence intensity.The overall fluorescence level of gastrical cancer cell MGC803 was lower than that of 293T(Figure 2), which may be due to high degree differentiation of MGC803,leading to an unstable expression level.

Usage and Biology

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 1
  Illegal XhoI site found at 21
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

References

BBa_K2295003 was sent in 2017 by Team Freiburg as an improvement of this BioBrick.

[1] HIF protein was rapidly and transiently stabilised at 3% and more at 1% oxygen tensions" Liu, Y. V., Baek, J. H., Zhang, H., Diez, R., Cole, R. N. and Semenza, G. L.(2007). RACK1 competes with HSP90 for binding to HIF-1alpha and is required for O(2)-independent and HSP90 inhibitor-induced degradation of HIF-1alpha. Mol. Cell25, 207-217