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<p> Our solution was to create a standardized bridging sequence that contained part types 1 through 5 to make the number of overall BsaI containing parts in the final reaction smaller. Instead of desgning primer that contain both BsaI and BsmBI sites on both ends, the 1-5 bridge only contains a BsaI sequence on the Type 1 Forward primer and on the Type 5 reverse primer. All other parts in the bridge contain only BsmBI sites. Therefore, when the parts are combined in a BsBI Golden Gate reaction, a single part to be used in BsaI assembly is formed.</P>
 
   
 
   
 
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<figcaption><b>Figure 1:</b> These are the results of Rice's one tube electroporation with <i>E. coli</i>.</figcaption>
 
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Revision as of 04:30, 16 October 2018


Integrated Human Practices

Optimization of Golden Gate Assembly

In order to increase the transformation efficiency of Golden Gate Assembly reactions we made standardized bridging sequences that reduced the number of parts in a reaction, increasing the likelihood of a successful assembly. Using this method, we were able to make many assemblies expressing different antibiotic resistance quickly.


Golden Gate Assembly is a molecular cloning method that contain modular, interchangeable parts. Parts are categorized intyo types according to their function. In order to make a full assembly plasmid that contains multiple parts of interest, such as a promoter of choice connected to a specific coding sequence and a specific origin, each part must first be put into a standardized entry vector. This entry vector is pYTK001, a GFP-dropout with a colE one origin of replication and a chloramphenicol resistance gene. Parts are made into transcriptional units by a first stage Golden Gate reaction that digests both the insertion sequence and the vector with BsmBI and ligates them back together. the end product is a self-replicating plasmid that acts to store the part for use in a larger assembly. The BsaI one sites on the insertion sequence are retained. This first stage transcriptional unit is termed a part plasmid


Multiple part plasmids are combined in a Golden Gate Assembly reaction. Each is digested with BsaI and ligated back together. However, the number of successful reactions, that is all part types ligating together to create the desired assembly, decreases as the number of parts increases. Therefore, larger, more sophisticated plasmids tend to be difficult to make.


Our solution was to create a standardized bridging sequence that contained part types 1 through 5 to make the number of overall BsaI containing parts in the final reaction smaller. Instead of desgning primer that contain both BsaI and BsmBI sites on both ends, the 1-5 bridge only contains a BsaI sequence on the Type 1 Forward primer and on the Type 5 reverse primer. All other parts in the bridge contain only BsmBI sites. Therefore, when the parts are combined in a BsBI Golden Gate reaction, a single part to be used in BsaI assembly is formed.

Fig 1. Schematic of reaction to make a 1-5 Bridge. The 1-5 Bridge reduces the number of parts in a BsaI Golden Gate assembly reaction to make a full plasmid that contains all part types.

Transformation of Assembly Plasmids into Mu Free Donors

Dr.Brian Redna of Gingko Bioworks emphasized that our kit would be limited by the ability of the plasmids to be inserted into the bacteria of interest as many can not be transformed with standard protocols. Therefore, we transformed the assembly plasmids into a strain of E. coli that can act as a plasmid donor in conjugations.

See Demonstrate Page

Figure 1: These are the results of Rice's one tube electroporation with E. coli.