contribution | SJTU-BioX-Shanghai

We have finished a report concerning the origin, impact, current treatment methods and recent research progress of heavy metal pollution. The report provides future teams (especially teams interested in dealing with heavy metal contamination in the near future) with a comprehensive review, which would accelerate the process of integrating background information and developing their projects. The report can also serve as an outline as well as an inspiration for more specific research activities in the future.

a) Inspiration for in situ microbial growth in a natural environment We designed a piece of hardware to solve the long-lasting problem of culturing engineered E. coli in a natural environment with appropriate chemical and physical conditions for bacterial growth, exposing the bacteria to the environment while guaranteeing biosafety. Not only can the hardware be suited for E. coli, but it also fills the gap between the genetically engineered machines and the application scenario for many former iGEM projects (and also projects to come).

b) Producing 3D-printed hardware pieces The 3D-printed pieces we designed provide useful structures and assemblies.

a) New application scenarios for OMVs
The outer membrane vesicle has been employed by the former iGEM teams. However, it has always been used as a mono-directional drug-delivery vehicle instead of a recyclable block for substance removal or accumulation. Therefore, our project provides a de novo application scenario for OMVs which may inspire future teams.

b) OMV production curve
We plotted an OMV production curve to characterize the change in OMV concentration with time (see Modelling), which is essential to the control and modification of OMV production. The future iGEM teams can build production models for their projects based on this knowledge.

The SpyTag/SpyCatcher system has been widely used by former iGEM teams in both intracellular and intercellular contexts. However, the system has never been adopted to generate cross-linkage between membrane vesicles, which is an efficient way of forming vesicle clusters and changing certain characteristics of the vesicles (see Results). Since membrane vesicles like exosomes and bacterial outer membrane vesicles are popular vehicles for various functions, our system would possibly inspire the design of even more adventurous parts.

Part	Description	Type	Registry
ClyA-PbrR-His6	Pb-binding protein, PbrR, is diplayed on the outer membrane by ClyA(also called HlyE, see BBa_K257002).Secreted OMVs with this will possess Pb(II)-binding acitivity. On the C terminus of PbrR we linked a 6 aa His-Tag, which can faciliate the recovery of the OMVs through nickel.	Composite Part	BBa_K4760000
SpyTag003-3xflag	This part is the reportedly improved SpyTag variant, SpyTag003, which can rapidly form isopeptide bond with SpyCatcher003.	CDS	BBa_K4760001
flexible GGGGS-His10 tag	This tag is designed under the idea that too large scaffold may mask the His6 tag, we used a higher-affinity his10 tag linked behind flexible linker GGGGS, which usually helps to avoid unfavorable secondary structure formation. Using this tag, we constructed the composite part BBa_K4760003.	Tag	BBa_K4760002
ClyA-PbrR-GGGGS-His10	This is an improved part of BBa_K4760000, using a flexible and higher-affinity linker BBa_K4760002 to replace the conventional His6 tag.	CDS	BBa_K4760003
Lpp'OmpA-His10	This is a variation of the reported Lpp'OmpA-His6 of Alves et al,1 possessing the same function of help recovering OMVs with nickel. The scaffold of Lpp'OmpA together with a flexible linker GGGGS would display a 10 aa His-Tag on the surface of Gram- bacteria outer membrane or OMVs, giving it Ni-binding affinity.	Composite Part	BBa_K4760004
Lpp'OmpA-SpyCatcher003	This is counterpart of Lpp'OmpA-SpyTag003. The two parts, expressed on two kinds of OMV surfaces, would bind with high affinity to each other, resulting in OMV crosslinking. The SpyCatcher we used is an optimized version improved by Keeble et al. 1	Composite Part	BBa_K4760005
SpyCatcher003	This is the counterpart of SpyTag003(BBa_K4760001). It would catalyze to form an isopeptide bond between itself and SpyTag003.	Composite Part	BBa_K4760006
Lpp'OmpA-SpyTag003	This is counterpart of Lpp'OmpA-SpyCatcher003. The two parts, expressed on two kinds of OMV surfaces, would bind with high affinity to each other, resulting in OMV crosslinking. The SpyTag we used is an optimized version improved by Keeble et al.1	Composite Part	BBa_K4760007
Lpp-OmpA-2xEAAAAK	Lpp-OmpA-2xEAAAAK is an improvement on Lpp'OmpA or Lpp-OmpA, a commonly used scaffold for bacteria outer membrane surface display. Addition of a rigid linker is thought to faciliate the display efficiency of smaller-size proteins of interest.	CDS	BBa_K4760008

Part

Description

Type

Registry

ClyA-PbrR-His6

Pb-binding protein, PbrR, is diplayed on the outer membrane by ClyA(also called HlyE, see BBa_K257002).Secreted OMVs with this will possess Pb(II)-binding acitivity. On the C terminus of PbrR we linked a 6 aa His-Tag, which can faciliate the recovery of the OMVs through nickel.

Composite Part

BBa_K4760000

SpyTag003-3xflag

This part is the reportedly improved SpyTag variant, SpyTag003, which can rapidly form isopeptide bond with SpyCatcher003.

CDS

BBa_K4760001

flexible GGGGS-His10 tag

This tag is designed under the idea that too large scaffold may mask the His6 tag, we used a higher-affinity his10 tag linked behind flexible linker GGGGS, which usually helps to avoid unfavorable secondary structure formation. Using this tag, we constructed the composite part BBa_K4760003.

Tag

BBa_K4760002

ClyA-PbrR-GGGGS-His10

This is an improved part of BBa_K4760000, using a flexible and higher-affinity linker BBa_K4760002 to replace the conventional His6 tag.

CDS

BBa_K4760003

Lpp'OmpA-His10

This is a variation of the reported Lpp'OmpA-His6 of Alves et al,1 possessing the same function of help recovering OMVs with nickel. The scaffold of Lpp'OmpA together with a flexible linker GGGGS would display a 10 aa His-Tag on the surface of Gram- bacteria outer membrane or OMVs, giving it Ni-binding affinity.

Composite Part

BBa_K4760004

Lpp'OmpA-SpyCatcher003

This is counterpart of Lpp'OmpA-SpyTag003. The two parts, expressed on two kinds of OMV surfaces, would bind with high affinity to each other, resulting in OMV crosslinking. The SpyCatcher we used is an optimized version improved by Keeble et al. 1

Composite Part

BBa_K4760005

SpyCatcher003

This is the counterpart of SpyTag003(BBa_K4760001). It would catalyze to form an isopeptide bond between itself and SpyTag003.

Composite Part

BBa_K4760006

Lpp'OmpA-SpyTag003

This is counterpart of Lpp'OmpA-SpyCatcher003. The two parts, expressed on two kinds of OMV surfaces, would bind with high affinity to each other, resulting in OMV crosslinking. The SpyTag we used is an optimized version improved by Keeble et al.1

Composite Part

BBa_K4760007

Lpp-OmpA-2xEAAAAK

Lpp-OmpA-2xEAAAAK is an improvement on Lpp'OmpA or Lpp-OmpA, a commonly used scaffold for bacteria outer membrane surface display. Addition of a rigid linker is thought to faciliate the display efficiency of smaller-size proteins of interest.

CDS

BBa_K4760008