Team:OUC-China/Parts

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Latest revision as of 02:04, 27 October 2012

Summary

Parts submitted:

33 Composite expression modules

10 Generator

4 Regulatory promoters

1 Ribosome Binding Sites

10 Coding proteins

10 RNA

Total: 68 Parts

All 68 parts have been submitted to the registry and can be found here.


Data for favorite parts

Gvp cluster(BBa_K737010) is a 1 kbp part contains 2 open reading frames coding for gas vesicle genes (gvpA gvpC-20) from Bacillus Planktothrix rubescens strain BC- Pla 9402. Promotion of the sequence results in expression of gas vesicles, organelles made entirely out of protein. These organelles contain gas and therefore provide bouyancy to the cell. This slows the rate of settling of the cells in different water layer from Lake Zurich by gas vesicles, see Walsby 2002.

the expression situation of the Gas vesicle gene cluster in Escherichia coli JM109, from the picture, we can see obviously gas vesicle due to the expression of bacteria produce clear separation phenomenon. The picture on the right of the experimental group were liquid surface layer and intermediate layer of fluid sampling results, we can see that the fluid layer surface bacteria volume density is obviously higher than that of the middle layer of fluid.

The picture shows the gas vesicle in the cells after expressed in the overall trend. We in the cultivation of the cells into logarithmic phase after transferring them to test tube, quiet place and culture 36 hours after using Trace stratified take liquid apparatus at the same time to each liquid layer for sampling. Then each layer of fluid samples of blood count the number of cells with statistics. Results show that gas vesicle due to the existence of the cell concentration in the test tube surface layer of fluid, and control group due to the action of gravity that most cells sedimentation in the test tube bottom.

Graph of OD measurements at 600nm of both experience group and control group. From the experimental results can be seen in the liquid surface .There cell number have significant abrupt transformation.


Constitutive GVPA Generator(BBa_K737006) It contains a constitutive promoter, GvpA protein , GFP protein and a terminator.This part can be used as a control of (J23106+P0412+R0011+B0034+GVPA+E0840). It can express GvpA protein and GFP constantly. When it transformed with (J23106+P0440+R0040+B0034+GVPC+E0840), the bacteria may can float with GFP!

YtfJ_Comparator( BBa_K737045) The leading sequence involved in the YtfJ transcript which can act as the target of the sRNA, spot42. It enables strong competition with the galk::GFP to interact with spot42 which shows great potential for constructing sRNA-mediated circuit.

This device mean to test feasibility of N/P comparator,replacing Nitrate,Phosphate inputs by IPTG,aTc。Small RNA Spot42 controls the GFP mRNA translation by blocking the RBS in galK.The galK and E0040 is fused together using ClaI site to avoid stop condon in standard scar.J23106 is a moderate promotor,and galK’s RBS is a moderate one.

Spot42 sRNA is under control of aTc inducible device,the buffer RNA YtfJ is under control of IPTG inducible device.

Constitutive GVPC Generator(BBa_K737007)It contains a constitutive promoter, GvpC protein , GFP protein and a terminator.This part can be used as a control of (J23106+P0440+R0040+B0034+GVPC+E0840). It can express GvpC protein and GFP constantly. When it transformed with (J23106+P0412+R0011+B0034+GVPA+E0840), the bacteria may can float with GFP!

Here is the result of SDS-polyacrylamide gel electrophoresis. We can see GFP and GvpC protein directly. Lane 4 is top10 control group, lane 3 is constitutive GvpA and GFP generator, lane 5 is constitutive GvpC and GFP generator. The upper white boxes are in the position about 26KD are GFP protein (25.8KD). The lower white box in the position of about 20KD is GvpC-20 (20KD). Because the MW(molecular weight) of GvpA protein is too small, it’s very difficult to see it on the SDS- polyacrylamide gel. But the expression of GFP suggests it may be expressed. Some papers say that GvpA is difficult to see is that GvpA is too hydrophobic to run out of sample hole.

Figure1.The result of SDS-polyacrylamide gel electrophoresis. Lane 4 is top10 control group, lane 3 is constitutive GvpA and GFP generator, lane 5 is constitutive GvpC and GFP generator. The upper white boxes are in the position about 26KD are GFP protein (25.8KD). The lower white box in the position of about 20KD is GvpC-20 (20KD).

Because the GvpA protein can’t be seen on the SDS-polyacrylamide gel electrophoresis, we wanted to use GFP to detect the expression of GvpA. We use a polycistron of gvpA(gvpC) and gfp to detect the RFU. We can see that, the RFU of Gvp and GFP Generator is about 2000, and the RFU of Gvp and GFP Generator is about 4000, while top 10 is about 0 as control. First, the RFU of these parts are rather high. Second, the E.Coli with these two plasmids can float. Third, we can see GvpC on the SDS-polyacrylamide gel. So, we can see the two parts can express Gvp Protein and work well.

Figure2.We can see that, the RFU of Gvp and GFP Generator is about 2000, and the RFU of Gvp and GFP Generator is about 4000, while top 10 is about 0 as control.

SrlA_Comparator(BBa_K737046)The leading sequence involved in the SrlA transcript which can act as the target of the sRNA, spot42. It enables strong competition with the galk::GFP to interact with spot42 which shows great potential for constructing sRNA-mediated circuit. This device mean to test feasibility of N/P comparator,replacing Nitrate,Phosphate inputs by IPTG,aTc.

Small RNA Spot42 controls the GFP mRNA translation by blocking the RBS in galK.The galK and E0040 is fused together using ClaI site to avoid stop condon in standard scar.J23106 is a moderate promotor,and galK’s RBS is a moderate one.

Spot42 sRNA is under control of aTc inducible device,the buffer RNA SrlA is under control of IPTG inducible device.

Spot42 (BBa_K737058)is a multitarget small RNA that mediates the discoordinate expression of the E.coli galactose operon and other sugar metabolic pathway,such as galK,nanC,ytfJ,srlA,which are the 5’ leader sequence of the corresponding metabolic enzymes.

Take galK for example,Spot42 causes translation repression by base pairing to RBS in the 5’ leader sequence,then block the recognition of the antiSD sequence on 30S subunit. It’s a very strong pairing(up to 20bases),but it only causes 2.6 fold repression according to Johannes H. Urban and Jo¨ rg Vogel,for it doesn’t result in the degradation of the RNA complex by RNaseE(ssRNA degradation) and RNaseIII(dsRNA degradation),both of them are major enzymes that causes RNA degradation in vivo.Spot42-galK complex degrades in a slow and presently unclear way. The base pairing is initiated by the recognition of seed region,which plays a significant role in the repression efficiency.

Spot42 have a weak Hfq binding site(whereas galK weaker),an endogenous terminator(unclear efficiency,maybe weak),and the multitarget repression stem-loop.


Data for optimized parts

galK:GFP generator(BBa_K737034) Compared with the original part, our new part possesses new function.Small RNA Spot42 controls the GFP mRNA translation by blocking the RBS in galK. The galK and E0040 is fused together using ClaI site to avoid stop condon in standard scar.J23106 is a moderate promotor,and galK’s RBS is a moderate one.

GFP generator(BBa_K737068) is provided by MIT in 2012 plates for teams. But we couldn’t transfer it successfully. So we constructed it again and submitted it to registry.

OmpR(BBa_K737067) Positively regulated, OmpR-controlled promoter ligated with B0034 can be used to control genetic circuit by light with light-dependent control element. This promoter is taken from the upstream region of OmpC. Phosphorylated OmpR binds to the three operator sites and activates transcription.

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