Characterization

We would like to characterize part BBa_K1894001, provided by 2016 iGEM team NANJING_NFLS. According to team NANJING_NFLS, BBa_K1894001 is a shuttle vector that can replicate in both E.coli and cyanobacteria, as it possesses the replication origin of both E.coli and cyanobacteria.  It was said to contain a CaMV35S promoter, multiple cloning sites (MCS) and rbcS polyA terminator, which makes it convenient to insert and express target gene and to screen out the recombinants. This plasmid is RFC10 compatible.

However, information such as whether BBa_K1894001 is a high copy plasmid, the transformation rate, or how the transformation of this shuttle vector affects the growth rate of E.coli and cyanobacteria remains unknown.

In order to provide more detailed information for future users of this shuttle vector, we designed two experiments:

1. To approximate BBa_K1894001 shuttle plasmid yield in different hosts
2. To quantify the number of E.coli cells after transformation of BBa_K1894001 shuttle plasmid
3. To quantify the colony yield after transformation of BBa_K1894001 shuttle plasmid

   Aside from transforming BBa_K1894001 shuttle plasmid into E.coli, we will also repeat the above experiments using transform shuttle plasmids PSB1c3, Pet-Blue2, Puc19. The data collected from these shuttle plasmids will be compared to the data collected from BBa_K1894001 shuttle plasmid transformants.

   We believe that this information is essential to user who would like to clone or express their DNA using this plasmid.

   Importance of our characterization:

   1. To determine whether BBa_K1894001 shuttle plasmid is a high-copy plasmid or a low-copy plasmid

      which allows users to decide whether this plasmid is suitable for their use

      • High-copy plasmids often result in a higher plasmid yield after plasmid purification, which is ideal for cloning or conducting downstream analysis.
      • High-copy plasmid is also favoured for gene expressions: In most cases, only one copy of the specific gene is placed on a plasmid. Therefore, the gene dosage depends only on the plasmid copy number. For the production of recombinant proteins, high-copy number plasmids are favoured because normally a high gene dosage leads to high expression levels¹.
      • Low copy plasmids are suitable for expressing toxic proteins
        1. To find out how BBa_K1894001 shuttle plasmid affects the growth rate of host cells
        2. Generally, plasmids of larger plasmids result in slower growth rates². This shuttle vector consists of 6913bp, relatively large compared to other cloning or expression vectors. Although this shuttle vector has a high-copy origin of replication, the size of the plasmid may hinder the growth rate. Characterization of colony yield and growth rate of transformants gives users what they should expect.
        3. If the growth rate of transformants is too low, it is not ideal for cloning nor is it for protein expression.
           1. Easier trouble-shooting for future users of the BBa_K1894001 shuttle plasmid

Design:

1. Construction of part BBa_K1894001 shuttle vector

BBa_K1894001 is a shuttle vector sized 6913bp. We could not get this part from the distribution kits, nor could we request it from the Registry. Therefore, we had to build this construct. As most DNA synthesis companies can only synthesis fragments of size ~3000bp, we decided to construct the plasmid using Gibson Assembly^® Master Mix and NEBuilder^® HiFi DNA Assembly Cloning Kit. These two DNA assembly methods are both scar-less. We divided the plasmid into 3 fragments (See Fig.1). Using primers, we added 24-25bp overlapping region to each fragment. We used both Gibson Assembly^® Master Mix and NEBuilder^® HiFi DNA Assembly Cloning Kit to assemble our fragments.

1. Characterization- DNA yield

   In order to determine whether BBa_K1894001 shuttle vector is a high-copy plasmid or a low-copy plasmid, we compared the plasmid yields of BBa_K1894001 shuttle vector, Psb1c3 vector, Pet-Blue 2 vector and PUC19 vector.

   We transformed the above plasmids into E.coli BL21 (DE3) and NEB5α. Same amount of DNA (1 ng) and competent cells are transformed. After one day of incubation at 37°C, colonies were picked and inoculated for 18 hours. To minimize DNA loss, plasmid purification was performed using Ethanol Precipitation.

Microcystis aeruginosa is one of the most common species responsible for harmful algal blooms. This cyanobacterium is capable of producing microcystin, a hepatotoxin that could damage the liver, and is regulated by United States Environmental Protection Agency. However, Microcystis aeruginosa has its ecological value of heavy metal sorption and oxygen synthesis. Through co-expression of a guide RNA and dead Cas9 enzyme (dCas9), we hope to silence the microcystin biosynthesis cluster (mcy), thus remove microcystin synthesis. Further investigations on whether the expression of dCas9 enzyme would affect the cyanobacterium’s heavy metal sorption rate and photosynthetic rate will also be carried out.

Notes