Part:BBa_K1333312

I13458-I13453-RBS-G3P(M13)

This is the expression vector we designed to express the G3P;, which is from M13 phage genome, with the regulation of araBAD promoter.

Introduction

G3P, a minor coat protein, is consisted by 406 amino acids, is about 42.5KDa. Different from other capsid proteins, G3P got a long hydrophilic region. G3P is not only necessary for the infectivity and stability of the phage, but also play an important role on stop assembling and the release of phage particles. As the M13 filament emerges from the infected cell, three to five copies of G3P are attached to the proximal tip of the bacteriophage particle. Also, G3P, which is associated with pVI at the proximal tip, is required for adsorption of the bacteriophage to the sex pili of new hosts and for penetration of the phage DNA.

We modified the M13 phagemid in several aspect. First, an antibiotic gene(Chloramphenicol or Kanamycin) has been inserted into LacZ locus for convenience of further selection. Second, select an insert site and insert the target gene. Third, knock out a crucial gene for the construction of rescue system.

The rescue system consist of two parts, the defective strain and the rescue plasmid. The defective strain is just as described above. And the rescue plasmid contains the crucial gene which is knocked out from M13 bacteriophage’s genome. An ideal rescue system can generate plenty of offsprings when two parts cotransferred into E.coli while few offspring can be obtained when either part transferred.

Since our project aims to construct a directed evolution system, M13 bacteriophage provide us with a small genome which can be modified easily. When integrated with bacteria two hybrid system, the rescue system will provide a selection pressure to push the evolution process.

Life Cycle

To better understand the crucial role M13 played in this system, an introduction of M13 life cycle is necessary. The general stages of M13 life cycle comprises: infection, genome replication, assembly of new particles, and then release of the offspring particles from the host. The genome replication is firmly connected with G2P and G5P. The single-stranded phage DNA that enters the cell is converted to a supercoiled, double-stranded replicative form (RF) by several host enzymes. Phage gene expression ensues, and G2P nicks the viral strand at the positive-strand origin. The 3’end of the nick is extended by DNA polymerase III, single-strand-binding protein and the Rephelicase. The displaced positive strand is recircularized by G2P and converted to RF DNA. Later, when sufficient G5P has accumulated, G5P dimers coat the single strands, earmarking them for assembly. Besides, G1P, G4P and g8p also play important roles in phage enveloping and release.

Function of other genes

G1P plays an important role in phage enveloping and release. G1P spans the inner membrane of M13-infected bacteria, interacts with the viral G4P and host-encoded thioredoxin and may also recognize the packaging signal in phage DNA, thereby initiating phage assembly.(BBa_K1333000)

The genome replication is firmly connected with G2P, The single-stranded phage DNA that enters the cell is converted to a supercoiled, double-stranded replicative form (RF) by several host enzymes. Phage gene expression ensues, and G2P nicks the viral strand at the positive-strand origin. The 3’end of the nick is extended by DNA polymerase III, single-strand-binding protein and the Rephelicase. The displaced positive strand is recircularized by G2P and converted to RF DNA.(BBa_K1333001)

G4P plays an important role in phage enveloping and release. GIV4P may form a gated channel connecting the bacterial cytoplasm to the exterior as a multimeric protein. It is synthesized in large quantities in infected cells, G3P interacts with G1P and is required for the induction of the E.coli psp(phage shock protein)operon that occurs during M13 infetion.(BBa_K1333003)

The genome replication is firmly connected with G5P. After the displaced positive strand is recircularized by G2P and converted to RF DNA, Later, when sufficient G5P has accumulated, G5P dimers coat the single strands, earmarking them for assembly. G5P binds strongly and in a cooperative manner to newly synthesized strands. The resulting G5P-DNA complexes move to specialized packaging sites on the bacterial membrane, where G5P is stripped from the viral DNA and is recycled into the cell. Also, G5P acts as a translational repressor by binding specifically to the leader sequences of viral mRNAs coding for gene II and other viral proteins.(BBa_K1333004)

G8P plays an important role in phage enveloping and release. G8P is one of the major capsid proteins, particles of M13 contain ≈2700 copies of the processed or mature 50 residue α helical protein, arranged in a cylindrical sheath around the bacteriophage DNA. About 2800 G8P could help with the synthesis of protein and it will be reserved at the host before the assembling of the phage.(BBa_K1333005)

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Expression Test in E. coli ER2738

araC (BBa_I13458), pBAD promoter (BBa_I13453) and an RBS (BBa_B0034) is constructed in front of the gene III (BBa_K1333002) in order, completing the G3P expression vector(BBa_K1333312) (Figure 1A).

Then, the plasmid is transformed into E. coli strain ER2738, which is a suitable host for M13 bacteriophage from New England Biolabs (https://www.neb.com/products/e4104-ecoli-k12-er2738), to test the expression efficiency.

Using arabinose to induce G3P to express, the expression level shows well relevance to the arabinose concentration (Figure 1B). Obviously, miscellaneous bands could be observed and the molecular weight of the signal is not as expected. However, compared with two groups of negative control, ER2738 only and ER2738 with M13KdZ (A M13 phage vector, encoding G3P without FLAG tag), it could be confirmed that the detected protein signal comes from BBa_K1333312.

Expression Test in E. coli ER2738 co-transformed with M13KdZdpIII

In our project (link to our wiki?), the BBa_K1333312 is mainly employed as a rescue plasmid, of which function is to rescue the capability to generate infectious progeny phage of M13KdZdpIII (Fig. 2A), a gene III defective M13 phage vector. So we tested whether it can be induced after co-transformation of two vector above.

By selecting different clones from the double-antibiotic plate to induce G3P expression, it can be known that different clones indeed perform differently while being induced (Fig. 2B).

For more information please visit our wiki

http://2014.igem.org/Team:SYSU-China/content.html#Project/Result/M13

family

with BBa_K1333001, BBa_K1333002, BBa_K1333003, BBa_K1333004, BBa_K1333005, BBa_K1333000