Golden Standard Tutorial

GS Recovery from the filter paper

To download the instructions for recovering plasmids from filter paper, click on the following button:
Recovery instructions

GS Wizard overview:





The tool is dividied in multiple sections. The upper part contains the TU to be selected by the user, clicking the color squares. The middle are is the blackboard where it is possible to select the receptor vector, the structure and the parts to create a construction. And the bootom part is the section with the multiple actions to do with the construction.
Note: This wizard allows the assembly of maximun of 6 TUs, the process to create a more complex assembly can be found in (Blazquez et al 2022)

Procedure

Step 1. Select a receptor vector

Golden Standard contains three possibilities, PUC, RK2 y pBRR1


Step 2. Structure selection. With zero, one or multiple tags.

The user can select the option to design a basic structure with promoter, RBS, cds and terminator or include tags in N-terminal or C-terminal position.



Based on the structure selection, the construction showed in the bottom panel is changing to acomodate the new parts.


2.1 Design

The first step is the TU selection clicking on the colored squares in the top section.



2.1.1 Custom level 1 constructions.

2.2.1 Select Golden Standard parts

- Click on a specific part (squared name) and select a element. It can be repeated as many times as elements to edit. The following image shows the output of the process after selection of the promoter Pem7, the RBS T7RBS, and so on. It is important to select the right parts based on their fusion sites, for example, if N-Tag is included, only a RBS with a BC fusion site is right. The tool shows all of them but only a RBS with a BC fusion site can give a right construction.




2.1.2 Select non Golden Gate parts

Previous requirements:

When a new sequence is included into the server some previous steps are required to perform a correct assembly.

New Sequence protocol:

1. If the assembly is basic (Promoter+RBS+CDS+Terminator):

Part

Position

modifications

Fixed sequences

Manual check point

Promoter

AB

Remove restriction enzymes

5'

GGTCTCAGGAG


3'

TACTAGAGACC


RBS

BD

Remove restriction enzymes

5'

GGTCTCATACT


3'

AATGAGAGACC


CDS

DG

Remove restriction enzymes

5'

GGTCTCAAATG

stop codon included

Remove ATG from sequence

3'

GCTTAGAGACC


Terminator

GI

Remove restriction enzymes

5'

GGTCTCAGCTT


3'

CGCTAGAGACC


2. If the assembly contains tags:

2.1 One N-tag:

The sequence require the following modifications, change the sequence according the type of the part.


Number of tags Part type Position modifications orientation Fixed sequences Manual check point

1 x N-Tag

Promoter

AB

5'

GGTCTCAGGAG


3'

TACTAGAGACC


RBS

BC

5'

GGTCTCATACT

Distance to ATG

3'

CCATAGAGACC


N-tag_1

CD

Remove ATG from original sequence

5'

GGTCTCACCATg

Reading frame

3'

gcAATGAGAGACC


CDS

DG

Remove ATG from original sequence

5'

GGTCTCAAATG

Reading frame

3'

GCTTAGAGACC

stop codon included




Terminator

GI

5'

GGTCTCAGGCT


3'

CGCTAGAGACC


2x N-tags

Promoter

AB

5'

GGTCTCAGGAG


3'

TACTAGAGACC


RBS

BC

5'

GGTCTCATACT

Distance to ATG

3'

CCATAGAGACC


N-tag_1

CD

Remove ATG from original sequence

5'

GGTCTCACCATg

Reading frame

3'

gcAATGAGAGACC


N-tag_2

DE

Remove ATG from original sequence

5'

GGTCTCAAATG

Reading frame

3'

tcAGGTAGAGACC


CDS

EG

Remove ATG from original sequence

5'

GGTCTCAAGGTcg

stop codon included

3'

GCTTAGAGACC


Terminator

GI

5'

GGTCTCAGCTT


3'

CGCTAGAGACC


1 x C-Tag

Promoter

AB

Remove ATG from original sequence

5'

GGTCTCAGGAG


3'

TACTAGAGACC


RBS

BD

5'

GGTCTCATACT

Distance to ATG

3'

AATGAGAGACC


CDS

DF

Remove ATG from original sequence

5'

GGTCTCAAATG

3'

TTCGAGAGACC

No stop codon




C-tag_1

FG

5'

GGTCTCACCATgc

Reading frame

3'

GCTTAGAGACC

Stop codon included

Terminator

GI

5'

GGTCTCAGCTT


3'

CGCTAGAGACC


2x C-tags

Promoter

AB

5'

GGTCTCAGGAG


3'

TACTAGAGACC

Distance to ATGHola

RBS

BD

5'

GGTCTCATACT


3'

AATGAGAGACC


CDS

DF

Remove restriction enzymes

5'

GGTCTCAAATG


Remove ATG from sequence

3'

TTCGAGAGACC


C-tag_1

FG

Remove restriction enzymes

5'

GGTCTCATTCGgc

Reading frame

Remove ATG from sequence

3'

GCTTAGAGACC


C-tag_2

GH

Remove restriction enzymes

5'

GGTCTCATTCGcg

Reading frame

Remove ATG from sequence

3'

GGTAAGAGACC


Terminator

HI

Remove restriction enzymes

5'

GGTCTCAGGTA



3'

CGCTAGAGACC


1 x N-Tag + 1x C-Tag

Promoter

AB

Remove restriction enzymes

5'

GGTCTCAGGAG



3'

TACTAGAGACC


RBS

BC

Remove restriction enzymes

5'

GGTCTCATACT

Distance to ATG


3'

CCATAGAGACC


N-tag_1

CD

Remove restriction enzymes

5'

GGTCTCACCATg

Reading frame

Remove ATG from sequence

3'

gcAATGAGAGACC


CDS

DF

Remove restriction enzymes

5'

GGTCTCAAATG


Remove ATG from sequence

3'

TTCGAGAGACC


Remove stop codon




C-tag_1

FG

Remove restriction enzymes

5'

GGTCTCATTCGgc

Reading frame

Remove ATG from sequence

3'

GCTTAGAGACC


Terminator

GI

Remove restriction enzymes

5'

GGTCTCAGCTT


3'

CGCTAGAGACC


2x N-tags + 1 C-tag

Promoter

AB

Remove restriction enzymes

5'

GGTCTCAGGAG



3'

TACTAGAGACC


RBS

BC

Remove restriction enzymes

5'

GGTCTCATACT

Distance to ATG


3'

CCATAGAGACC


N-tag_1

CD

Remove restriction enzymes

5'

GGTCTCACCATg

Reading frame

Remove ATG from sequence

3'

gcAATGAGAGACC


N-tag_2

DE

Remove restriction enzymes

5'

GGTCTCAAATG

Reading frame

Remove ATG from sequence

3'

tcAGGTAGAGACC


CDS

EF

Remove restriction enzymes

5'

GGTCTCAAGGTcg


Remove ATG from sequence

3'

TTCGAGAGACC


C-tag_1

FG

Remove restriction enzymes

5'

GGTCTCATTCGgc

Reading frame

Remove ATG from sequence

3'

GCTTAGAGACC


Terminator

GI

Remove restriction enzymes

5'

GGTCTCAGCTT


3'

CGCTAGAGACC


1 x N-Tag + 2 x C-Tag

Promoter

AB

Remove restriction enzymes

5'

GGTCTCAGGAG


3'

TACTAGAGACC


RBS

BC

Remove restriction enzymes

5'

GGTCTCATACT

Distance to ATG

3'

CCATAGAGACC


N-tag_1

CD

Remove restriction enzymes

5'

GGTCTCACCATg

Reading frame

Remove ATG from sequence

3'

gcAATGAGAGACC


CDS

DF

Remove restriction enzymes

5'

GGTCTCAAATG


Remove ATG from sequence

3'

TTCGAGAGACC


Remove stop codon




C-tag_1

FG

Remove restriction enzymes

5'

GGTCTCATTCGgc

Reading frame

Remove ATG from sequence

3'

GCTTAGAGACC


C-tag_2

GH

Remove restriction enzymes

5'

GGTCTCAGCTTcg

Reading frame

Remove ATG from sequence

3'

GGTAAGAGACC


Terminator

HI

Remove restriction enzymes

5'

GGTCTCAGGTA


3'

CGCTAGAGACC


2 x N-Tag + 2 x C-Tag

Promoter

AB

Remove restriction enzymes

5'

GGTCTCAGGAG


3'

TACTAGAGACC


RBS

BC

Remove restriction enzymes

5'

GGTCTCATACT

Distance to ATG

3'

CCATAGAGACC


N-tag_1

CD

Remove restriction enzymes

5'

GGTCTCACCATg

Reading frame

Remove ATG from sequence

3'

gcAATGAGAGACC


N-tag_2

DE

Remove restriction enzymes

5'

GGTCTCAAATG

Reading frame

Remove ATG from sequence

3'

tcAGGTAGAGACC


CDS

EF

Remove restriction enzymes

5'

GGTCTCAAGGTcg


Remove ATG from sequence

3'

TTCGAGAGACC


Remove stop codon




C-tag_1

FG

Remove restriction enzymes

5'

GGTCTCATTCGgc

Reading frame

Remove ATG from sequence

3'

GCTTAGAGACC


C-tag_2

GH

Remove restriction enzymes

5'

GGTCTCAGCTTcg

Reading frame

Remove ATG from sequence

3'

GGTAAGAGACC


Terminator

HI

Remove restriction enzymes

5'

GGTCTCAGGTA


3'

CGCTAGAGACC



Once, the sequence is corrected, it can be included in the system.



  1. Click the combo-box and select “Custom part”. A pop up window is opened and it can be fill with the custom name, the sequence or the size of the sequence and the concentration. The following figure shows the case where the cds is selected.



b) Click on Submit button to save the changes.



Step 3. Set up calculations


After the selection of all parts, the final information related to final concentration, enzyme requirements, required water and buffer quantity can be calculated clicking on the button “Calculate set-up“






Step 4. See the results


The construction can be stored as genbank format clicking on the Download Genbank Button, or can be viewed by the viewer tool or it can be set up.




Step 4.1 Setup

The concentrations and sequence lengths form the assembly are used to calculate the final protocol parameters. The Golden Gate Wizard tool send the information to Golden Gate Setup module and redirect to Golden Standard Setup page.




A table with the information introduced previously appear, with the final calculations. The results can be easily edited clicking on the squares. Initially, the concentrations are empty and it is necessary to fill them to have final concentrations instead infinity values. In the upper part of the table the total amount of volume or the number of desired fmol can be modified if the user needs.





After the calculation is done, this table can be printed using the button Print.



Step 4.2. View the construction




To store the construction, it can be downloaded clicking the Export button. The server will redirect to the vector viewer (Open-vector-editor) showing the final construction. By default Single cutters restriction sites are activated, but to show the restriction sites of the enzymes used in Golden Standard it can be activated Clicking on .
A menu will open with the activated enzymes. And to include others, clicking on the top-right triangle a list of them will be shown. GoldenStandard can be selected and it will be included into the activated enzymes.




Step 4.3. Download the construction

To download the construction in Genbank format, it is necessary click on Download genbank button.