Team:SJTU-BioX-Shanghai/result

Result overview

After one month of brainstorming and three months of hard lab working, the SJTU-BioX-Shanghai team has successfully submitted 13 new parts, of which a large portion, such as [http://partsregistry.org/Part:BBa_K387003 PfdhF promoter],[http://partsregistry.org/Part:BBa_K387001 MEF2-JeT promoter] and [http://partsregistry.org/Part:BBa_K387012 CRE-JeT promoter], are new parts that are demonstrated capable of working as expected. Moreover, [http://partsregistry.org/Part:BBa_I716211 RNase barnase] is an existing part in the Partsregistry, and we also have it characterized in order to provide more data.

Furthermore, we have built models which correspond to most of our experiment results, meaning they are highly simulative and thus capable of providing essential support for our design.

With the brilliant results of these main modules, our project, Synthetic-biological approaches to Osteoarthritis was proven to be highly feasible and has a great prospect to be further studied and applied. Now let us take a look at the experiment outcomes.

Parts

Go to [http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2010&group=SJTU-BioX-Shanghai Partsregistry] to see our submitted parts.

Demonstration of some parts we submitted

Eukaryotic approach

Channelrhodopsin-2 (ChR2)

ChR2 is a widely used membrane-cross molecule in neuron-related experiments. When illuminated, it could be penetrated for many cations, such as sodium ion(Na+), calcium ion(Ca2+) and even some organic molecules which are smaller than the hole formed by ChR2. ChR2 is sensitive, especially to the light ranging in a particular bandwidth, usually between 450nm-470nm and this is the region for blue light.

Structural test

• Idea: To detect whether ChR2 is correctively expressed cross the membrane of another kind of cell, especially in mammalian somatic cells, we could transfect 293T cells with pcDNA3.1 containing the gene encoding ChR2-eYFP. If it could be expressed as the theory told us, the fusion protein--ChR2 and eYFP, will be embedded in the membrane.

• Method: We have prepared coverslips in the wells of cell culture plate before adding 293T cells. The medium is composed by DMEM, 10% FBS and 1% Penicillin/Streptomycin. We made transfection on the next day. After another 24h-culture, we picked up the coverslips out of the wells and washed them carefully by PBS. Then we treated them with 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride, which is known as DAPI dihydrochloride. This reagent could stain cell nuclear into blue, so it would be easy to observe with the help of microscope.

• Result: As we expected, we find the blue points which represent cell nuclears are surrounded by green outer covering under Laser Scanning Confocal Microscope (Leica), as shown below. After reading the paper published by Stanford on Nature Neuroscience in 2005 (Millisecond-timescale, genetically targeted optical control of neural activity), we could tell the green-labeled membrane has ChR2-eYFP embedded in. From the photo, we can be sure that ChR2 is expressed in 293T cell and more importantly, it has its correct position in the membrane, which is an essential precondition for its function. At this point, we proved the structural correctness of ChR2.

Function

• Idea: Since ChR2 could be expressed structural correctively in another kind of cell, we start to seek for the answer of whether it could work normally as in neurons. As ChR2 could initiate calcium influx, we could test its function by examining the transcription level of some certain genes which are sensitive to the change of Ca2+ concentration. Luckily, we found Zif268, a gene in Mouse Genome, and it would be up-regulated when meeting with an increase of Ca2+. On the other hand, we found discontinuous illumination is better for ChR2’s penetration for cations, while continuous illumination could cause inactivation of ChR2.

• Method: We cultured ECHO cells in 35mm-plates and the medium is composed by Ham’s/F12, 10% FBS, 1% Penicillin/Streptomycin and 0.25% Zeocin. After 24 hours, we transfected ECHO cells with pcDNA3.1, the same as above, containing ChR2-eYFP, and we wrapped the plates with foil. 24h after transfection, we carried the plates into darkroom for later experiments. We used a red light source to provide enough light for us to see and blue light pulses (around 460nm) are used to illuminate the cells. Since the blue light source is continuous, we got light pulses by pressing the switch button in a given rhythm. We tested different timescale to see the change of Zif268 transcription level. Besides the semi-quantitative measurement, we later led Realtime PCR to test quantitatively.

• Result: According to the different conditions of experimental and control groups, we could see that RNA extraction was successful. From the RT-PCR results, we know the cells transfected by ChR2-eYFP (pcDNA3.1) then illuminated for 30min showed the brightest band in Zif268 expression, followed by transfected/illuminated (20min), transfected/no illumination and no transfection/no illumination. While the bands of GAPDH showed no difference among them. The pictures are below.

Real-time PCR

In order to render the result more convincing, we also conducted Real-Time PCR for these two genes.

(blank)

GDF5 promoter

As can be seen from the figures (taken via fluorescent confocal microscopy), ChR2-eYFP expression is much higher in C3H10 cell line than in ECHO, suggesting that GDF5 promoter activity is much higher in mesenchymal stem cell-C3H10 cell line. Considering the transfection rate in C3H10 cell line is not as high as that in ECHO cells, the difference in activity of GDF5 promoter in these two cell lines could be much greater than what is shown in the figures. Studies have demonstrated that GDF5 gene is involved in joint formation and it is expressed in the regions of future joints during early development. Meanwhile, C3H10 cell line which is generated from mesenchymal stem cell is supposed to involve in embryonic differentiation of joint. Our result can be strong enough to show that a tissue specific promoter has been constructed successfully.

MEF2-JeT promoter

(blank)

CRE-JeT promoter

(blank)

Prokaryotic approach

PfdhF, the hypoxia-inducible promoter

Yupeng He built and tested the hypoxia-inducible promoter, fdhF promoter (PfdfF, [http://partsregistry.org/wiki/index.php?title=Part:BBa_K387003 Part:BBa_K387003]). In this test experiment, we expected to see that, at the same OD₆₀₀, the activity of PfdhF is higher in hypoxia than in normal oxygen pressure (20%). At the beginning, both the hypoxia group and control (20% oxygen partial pressure, i.e. in air) group were incubated with 200 microlitre suspension of bacteria with the testing part (PfdhF followed by firefly luciferase). Then, we sampled both group and measured OD₆₀₀ and value of luciferase every 30 minutes (control group) or 1 hour (hypoxia). The figure was the result of this test. Our result showed that this part worked as we expected: under hypoxia, the efficiency of this promoter was much higher than that under the condition of 20% oxygen at the same OD₆₀₀. (One thing that needs to be noticed is that at 0.4 OD₆₀₀, the bacteria are in log phase and the PfdhF begins to work.)

Then we wanted to know the difference of PfdhF activity under these two conditions. So, we plotted another figure with OD₆₀₀ as x axis and the ratio of PfdhF activity under hypoxia to that under normal oxygen pressure as y axis. This curve showed that, during and after the log phase (OD₆₀₀>0.4), the activity of PfdhF is over 1.5 times more under hypoxia than under normal 20% oxygen pressure. Particularly, this ratio is over 3 when OD₆₀₀>0.5. Furthermore, in the normal 20% oxygen group, as the OD₆₀₀ increases, the oxygen supplies may be insufficient for E. coli which may lead to the increase of PfdhF activity. In this case, the activity of PfdhF under normal condition is probably overestimated. With all these data and analysis, it is evident that PfdhF is a rather good hypoxia-inducible promoter with much higher activity under hypoxia than under 20% oxygen pressure.

Characterization

RNase barnase

Due to our design containing the RNase barnase as part of our prokaryotic Supervisor, we decided to go on a journey to characterize the [http://partsregistry.org/Part:BBa_I716211 part].