Team:SUSTech Shenzhen/Composite Part

Composite Parts

Contribution

This year, we constructed 12 composite Parts, including functional pigment proteins and fluorescence proteins driven by bacterial promoters. Our aim is to construct plasmids express proteins that can be seen by naked eyes without special devices, and thus are friendly to all labs without limitation on conditions. More importantly, our collections are convenient to use as backbones for construction of BioBricks and other plasmids.

1. Chromoprotein collection

We successfully constructed 8 plasmids with various pigment coding sequence, including eforRed, gfasPurple, Fw Yellow and SpisPink.

Name	Description	Length	Feature
<partinfo>BBa_K1943000</partinfo>	fwYellow, yellow chromoprotein reporter system (Strong Promoter, Strong RBS)	912bp
<partinfo>BBa_K1943001</partinfo>	fwYellow, yellow chromoprotein reporter system (medium Promoter , weak RBS)	914bp	221x67px
<partinfo>BBa_K1943002</partinfo>	gfasPurple, purple chromoprotein reporter system (Strong Promoter, Strong RBS)	867bp	224x58px
<partinfo>BBa_K1943003</partinfo>	gfasPurple, purple chromoprotein reporter system (medium Promoter , weak RBS)	869bp	227x58px
<partinfo>BBa_K1943004</partinfo>	eforRed, Red chromoprotein reporter system (Strong Promoter, Strong RBS)	879bp	204x58px
<partinfo>BBa_K1943005</partinfo>	eforRed, Red chromoprotein reporter system (medium Promoter , weak RBS)	881bp	217x62px
<partinfo>BBa_K1943006</partinfo>	spisPink, Pink chromoprotein reporter system (Strong Promoter, Strong RBS)	876bp	214x57px
<partinfo>BBa_K1943007</partinfo>	spisPink, Pink chromoprotein reporter system (medium Promoter , weak RBS)	878bp	212x55px

Compared to the common used fluorescence protein such as GFP, RFP, chromoproteins are obviously more useful reporter genes. In our parts, the coding sequences of these pigments are all among 700bp long, and the expression efficiency is high according to our experiment results.

We contributed this series of the chromoprotein plasmid for the reason that they have significant advantages to be used as backbones. The length of 700bp is easy to be distinguished from the original backbone (around 2000bp) by gel electrophoresis when constructing new expression system. Additionally, this kind of backbones are superior because we are able to directly observe the expression results by using naked eyes. It will be much more convenient for the laboratory with limited conditions and also save our time. In the tests, the color of colonies were easy to recognize after 18 hours of incubation.

Our plasmid can also be a substitute for J04450, which kindly recommended to be used in plasmid construction by replacing the RFP gene. For the same reason, we provide these pigment sequence to make the construction process more efficiently.

The expression of 8 plasmids.

Source

The following list is the BioBricks we’ve used in construction and their detailed design information.

Part	Type	Description	Backbone	Location on the plate
<partinfo>BBa_K880005</partinfo>	Promoter + RBS	Strong Promoter + Strong RBS	pSB1C3	3F 2016 Kit Plate 2
<partinfo>BBa_K608007</partinfo>	Promoter + RBS	Medium Promoter + Weak RBS	pSB1C3	5G 2016 Kit Plate 1
<partinfo>BBa_K592012</partinfo>	Chromoprotein	eforRed, red chromoprotein	pSB1C3	15I 2016 Kit Plate 6
<partinfo>BBa_K1033919</partinfo>	Chromoprotein	gfasPurple, purplr chromoprotein	pSB1C3	9K 2016 Kit Plate 6
<partinfo>BBa_K1033910</partinfo>	Chromoprotein	fwYellow, yellow chromoprotein	pSB1C3	6K 2016 Kit Plate 4
<partinfo>BBa_K1033932</partinfo>	Chromoprotein	spisPink, pink chromoprotein	pSB1C3	11K 2016 Kit Plate 6
<partinfo>BBa_B0015</partinfo>	Terminator	Double terminator	pSB1C3	3F 2016 Kit Plate 3

2. sfGFP collection

We submitted this part of super folder GFP (sfGFP) with a strong promoter J23116. Under this expression system, a very bright blue-green fluorescence will be observed after 6 hours of incubation. Its excited wavelength is close to the UV light. We commonly manipulate it by using the UV in super clean bench. The fluorescence is strong enough to be observed by naked eyes. For this reason, sfGFP is always used as reporter gene under the plasmid construction.

This plasmid can be used as a vector. After ligation, it is easy to pick up the single colony without blue-green fluorescence.

On the other part we submitted mutated sfGFP (msfGFP). It has one base-pair difference with the original sfGFP on its 165th site. We changed the base pair from AT to GC by using whole plasmid mutagenesis to remove KpnI restriction site. We kindly hope the little effort can bring convenience to the following iGEMers.

In addition, we also constructed the sfGFP expression system with a terminator following the coding sequence. While the two groups (with and without terminator) showed no significant difference in our experience. We hope these parts can be provided as comparable experimental groups to quantify the efficiency of bacterial terminators.

The expression of mutated sfGFP (the middle and right tube) under UV.

Name	Description	Length	Feature
<partinfo>BBa_K1943008</partinfo>	Strong promoter+ strong RBS+ sfGFP	778bp	133x52px
<partinfo>BBa_K1943009</partinfo>	Strong promoter+ strong RBS+ sfGFP+ terminator	918bp	206x49px
<partinfo>BBa_K1943010</partinfo>	Strong promoter+ strong RBS+ msfGFP	781bp	145x50px
<partinfo>BBa_K1943011</partinfo>	Strong promoter+ strong RBS+ msfGFP+ terminator	915bp	215x50px

Made by from the iGEM team SUSTech_Shenzhen.

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• ©2016 SUSTech_Shenzhen