Part:BBa_K3790232

T7 gene product 5.7 driven by pBAD/araC

Introduction

In order to turn the host bacteria into ‘factories’ that concentrate on manufacturing parts of the virus, some phages have the ability to shut off the endogenous transcription of host bacteria. As for T7 phage, there are three gene products collaboratively realizing the function, which are gene product (gp) 0.7, gp2 and gp5.7.

Gp0.7 and gp2 are early phage genes, which means they are transcribed by the host's RNA polymerase at the very beginning of the infection process.

Gp0.7 is a protein kinase without strong specificity. Some studies shown that since it can phosphorylate RNAP of E. coli., it can somehow help inhibit the transcription, while others mentioned that there might be unknown mechanisms. For now, the function of gp0.7 is complicated and not well studied. It can interfere with transcription somehow, but the effect is not clear.

Gp2 is a small protein that can bind to both the 1.1 domain of σ70 factor and the β’ subunit of the host's RNAP, results in effective inhibiting of the transcription initiated by σ70-RNAP complex.

However, in stressful conditions, such as growth plateau stage, E. coli will mainly use σS factors to initiate transcription, which makes gp2 less effective to shut off transcription. Therefore, gp5.7 is required for phage to effectively infect E. coli as growth plateau. Gp5.7 is coded by a mid-stage phage gene. It binds to σS factors and RNAPs of E. coli, to inhibit the transcription initiated by σS-RNAP complex.

Usage and Biology

Flexible growth inhibition

σS factors are typically responsible for genes concerning adaption to suboptimal growth conditions, and only take dominance when the environment is stressful. However, σS factors actually exist at any condition (it’s the matter of amount), and are essential for cell growth. In E. coli genome, several essential genes for biofilm formation and metabolism are regulated by σS factors. Therefore, by inhibiting the transcription initiated by σS-RNAP complex, though may not as effective as gp2, gp5.7 still can hinder cell growth. Thanks to its lack of effectiveness, unlike gp2, the effect of gp5.7 on inhibiting cell growth is notably related to its expression level, thus can engineered using various inducible promoters.

Expression regulation

Gp5.7 can effectively inhibit σS promoters, showing the potential to serve as an expression regulator in gene circuits. It has another advantage that its regulation can work from a single σS promoter element, not relying on other protein binding region on DNA sequence. That means we can freely engineer other regulating elements to the system and make it regulated by several factors. This feature is important in designing highly complex logical gene circuits. We believe that expression regulation using gp5.7 can be very useful in the field of synthetic biology.

Make the protein of interest purer in dense culture

In our project, we aim to directly use E. coli lysate for LAMP reaction, which can save the cost typically spent for protein purification, storage and transporation of purified proteins. LAMP is catalyzed by Bst DNA polymerase. One major challenge is that, there’re many background expressed proteins in the cytoplasm which may influence the activity of Bst enzyme. So, we want our protein of interest purer in the lysate. One way to make this happen is to inhibit the expression of all other proteins except the protein of interest.

By using gp2, we can easily inhibit the expression of most of the proteins in E. coli, but the expression of gp2 must happen during exponential growth stage. Only during that stage, σ70 factors are the major σ factor regulates all the transcription. However, expression of gp2 will immediately stop the growth of the bacteria, reduce the density of cell, resulting a very low total protein yield, which will also decrease the activity of the enzyme.

In contrast, gp5.7 can effectively inhibit almost all the transcription when growth plateau, during which the density, that could be measured by OD600, of bacteria has reached the up-limit. We hypothesize that using gp5.7 can lead to purer and larger amount of protein of interest in the final lysate.

Design

Gp5.7 coding sequence synthesis

Since the length of gp5.7 coding sequence is approximately 200bp, we chose to synthesize the sequence ourselves by Oligo assembly using Phanta polymerase. We obtained the sequence information from NCBI and designed synthetic primers for synthesis.

Also, we need the coding sequence of gp2, which can also be obtained by Oligo assembly.

Figure 1. Oligo assembly by PCR. It is generally used to construct completely new or special-purpose DNA. This method may have the disadvantage of a high mutation rate when operated. Once, we had to sequence nine clones of the same construct to get a single correct one. The reason for this is most likely due to complex annealing and amplification. We suggest to have 10-15 rounds amplification without F1 or R1 primer, then add those two primers to have another 25 rounds. Must use high-fidelity enzymes for this method. Due to the pricing, we always use 60bp primers, 58 overlapping annealing temperature, to assemble 300-500bp DNA fragment.

The length of gp5.7 and gp2 coding sequence are respectively 210 bp and 195 bp.

Figure 2. Assembled coding sequences, gp2 and gp5.7. The first lane was loaded with DNA ladder, sizes were marked on the image. The brightest band of 750 bp was about 100 ng, and other bands about 50 ng. Lanes with correct sized amplified DNA were labeled. After PCR cloning, several bacterial clones were picked, grew into cultures and sent for Sanger sequencing. Then, we verified the sequencing results, and used the correct ones for further experiments.

Toxin expression strategies

Gp5.7 can be regarded as a kind of toxin to E. coli. One challenge of expressing toxin is decreasing the basal expression, or in another word, decreasing the leakage. We chose pBAD, which is a typical inducible promoter with low level of basal expression. Furthermore, we cloned this part into a low-copy or medium-copy vector in order to further reduce the basal expression of gp5.7. In our experiments, we used a vector with p15A ori replication origin, which is a medium-copy replication origin.

Characterization

The four dual-plasmid systems shown below are respectively transformed into DH5α E. coli. Three colonies of each strain were picked out and cultivated in 37℃ overnight. The bacteria solution was diluted to 5% by LB culture medium with chloramphenicol. After 5 hours of cultivation in 37℃, different concentrations of arabinose (inducer to pBAD) were added, and OD600 and fluorescence intensity (excitation: 488nm, emission: 530nm) were measured every 30 minutes. We stopped samples on ice before measure them using a 96-well plate reader.

Figure 3. The two plasmids connected with dual-arrow-line were co-transformed into one bacteria, selected and maintained by two antibiotics. Please check https://parts.igem.org/Part:BBa_K3790232 where Figure 4 and 5 verified our promoters are indeed σS and σ70 promoters.

Growth inhibition

Figure 4. The growth of bacteria with dual-plasmid combination 2 shown in Figure 3. In our experimental settings, 1 OD equals to 10^8 950-nm diameter silica nanoparticles made by NanoCym.

From Figure 4, we can see a clear suppression of bacteria growth after gp5.7 inducation, and the extent of growth inhibition is relevant to induction level. Interestingly, the growth of inhibition doesn't last long, cells in all the groups soon recover their growth rate (we successfully model this, more details at https://2021.igem.org/Team:Fudan/Model ).

Expression inhibition

Figure 5. The GFP per OD measured during growth of bacteria with dual-plasmid combination 2 in Figure 3. The GFP is σ70 driven.

Figure 6. The GFP per OD measured during growth of bacteria with dual-plasmid combination 3 in Figure 3. The GFP is σS driven.

From Figure 5 and 6, we can see that gp5.7 can effectively inhibit GFP expression driven by a σS promoter. It can also slightly inhibit expression driven by a σ70 promoter. Surprisingly, both trends are similar.

Compared with gp2

Figure 7. Different groups' GFP concentration/OD after 8 hours of cultivation under different induction levels are compared.

Figure 8. Different groups' GFP concentration/OD after 8 hours of cultivation under no induction are compared.

From Figure 7, we can see that both of gp2 and gp5.7 can inhibit σ70 and σS simultaneously. Gp2 shows stronger ability to inhibit σ70, while gp5.7 shows stronger ability to inhibit σS, which confirms to expectation.

What's worth mentioning is that, Figure 7 can't indicate that gp5.7 has stronger inhibition ability then gp2. From Figure 8 we can see that the protein expression has already been greatly inhibited in strain with pBAD driven gp2 even there's no arabinose induction. It suggests that the leakage expression of gp2 is enough for marked transcription inhibition. In the main page of BBa_K3790231, we further demonstrate that different induction levels can't notably influence the effects of pBAD driven gp2. Therefore, pBAD driven gp5.7 can better serve our needs of positively triggering the transcription shutoff in E.coli..

For more details about this part, please check out our presentation video.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 1205
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 1144
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 979
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 961