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Objective

Figure 1: Assay Plasmids' cassettes.

There are two parts in our system, the Phage and the E. coli. Before we can test them together, we have to make sure that our parts work individually. For that purpose, we allocated two team members to create assays that can test the function of our E. coli parts, the inducible RNA polymerases, and characterize them.
The cassettes that are used in the plasmids are shown in Figure 1.

Work plan

We intend to create two colored assays that can indicate and quantify the expression of the different polymerases from our plasmids. Each assay gene will be cloned after each of the RNA polymerase promoters, so that the expression of the polymerases will be linked to the color developed. Before using them with the designated RNA polymerase plasmid, we intend to characterize them by using different inducer concentrations and measuring the absorbance.
Main Parts:

1. Alkaline Phosphatase Assay: In E. coli, alkaline phosphatase (ALP) is encoded by the phoA gene. The wild type phoA gene from Citrobacter has a signal sequence directing export of alkaline phosphatase into the periplasm, where it is active. In this protocol, the colorless substrate p-nitrophenyl phosphate (PNPP) is hydrolyzed by ALP, producing nitrophenol, a yellow color product, which is quantified by an assay at a maximum absorbance 420 nm.
2. xyIE Assay: The xylE gene from Pseudomonas putida TOL pWW0. This gene encodes the enzyme catechol-2,3-dioxygenase (metapyrocatechase), which converts catechol to the bright yellow product 2-hydroxy-cis,cis-muconic semialdehyde. This is a useful reporter gene; colonies or broths expressing active XylE, in the presence of oxygen, will rapidly convert catechol, a cheap colorless substrate, to a bright yellow compound with an absorbance maximum around 380 nm.
3. Promoters: In order to check as many different polymerases to see with which set of enzymes we achieve minimum cross-reactivity, we worked with 5 polymerases. Both the polymerases and their matching promoters were acquired from Christopher A. Voigt1. He sent us the four RNA polymerases promoters: pT7*,p T3, pK1F, and pN4 with a T7 terminator at the end. These four plasmids will also function as our backbone plasmids, and to them we'll clone the assay genes along with their RBS. The Sp6 promoter was added via PCR over the T7 promoter.
4. Controls:
1. Positive- In order to see whether our assay's genes, phoA and xyIE, work properly and generate a colored output, we created a positive control. The positive control will consist of a Tetracycline repressible promoter (pTetO) before each of the assay's genes. In the absence of an inducer, TetR binds to tetO and prevents transcription. It can be turned on when an inducer, such as tetracycline, binds to TetR and causes a conformation change that prevents TetR from remaining bound to the operator.
   

   

   Figure 2: an illustration of the pET system as designed originally [A] and our own pET as used in our experiment [B]
   When the operator site is not bound, the activity of the promoter is restored. Unfortunately, we don't have a bacterium strain with the repressor we plan to use the pTetO as a constitutive promoter.
2. Negative: We have two kinds of negative controls: The first one is to see whether we get any absorbance without the presence of the assay parts, promoter + assay gene. The second one is to measure the leakage of the polymerase promoters. For that reason, we will measure the absorbance of the promoter plasmids without the polymerase genes. 
5. pET Expression System: In order to characterize our assay, we intend to use the pET expression system. The host cell for the pET expression system is a bacteria which has been genetically engineered to incorporate the gene for T7 RNA polymerase, the lac promoter and the lac operator in its genome (BL21 strain). When lactose or a molecule similar to lactose is present inside the cell, transcription of the T7 RNA polymerase is activated. Therefore, we have a similar system to the T7 RNA polymerase plasmid that we intended to create in the first place. By transformation of our pT7 test plasmid to the engineered bacteria with the endogenous T7 polymerase gene, we can characterize our pT7 test plasmid. By adding rising concentrations of the inducer, IPTG, and measuring the absorbance for each one, we can deduce whether we have a rising concentration of our assay genes, phoA and xyIE. However, this only works in bulk or culture assay and not single cell assays, since in single cells the lac system is on/off. Meaning that at low concentrations of IPTG some cells will turn on, while the rest will be off. An illustration of the normal pET system and our "made" system is shown in Figure 2.

Results

The Assay Genes

Figure 3: gel electrophoresis for the PCR results of all the promoter backbone plasmids. Expected size: 3400 bp.  The left lane is a standard 1 Kb ladder (NEB). A- Only the T7 promoter yielded positive results. B- Only the K1F promoter yielded positive results. C- the remaining promoters- T3,N4 and SP6 yielded positive results.

In order to have our assay ready we ordered the Alkaline Phosphatase reporter gene, phoA, from the registry (BBa_J61032). We already had the xyIE reporter gene in the distribution kit (BBa_J33204). We added XhoI and XmaI restriction sites in both ends of the parts via PCR, for the phoA gene we also added an RBS (BBa_B0064). Since the phoA sequence was reported to be inconsistent we sequenced it and found that there was only one silent mutation in the phoA CDS.

The Promoter Plasmids

Due to the fact that the promoter plasmids had an mRFP gene between the promoter and the terminator, we ran another PCR in order to delete it, in which the primers were at both ends of the mRFP facing outwards. In order to have a fifth plasmid with the SP6 promoter2 we performed another PCR, only with different primers: we engineered primers that had the Sp6 promoter in them while the template was an pT7 plasmid. This way, the T7 promoter was replaced with the Sp6 promoter. In all of the reactions described above, we added XhoI and XmaI restriction sites in both ends of the plasmids.
As shown in Figure 3 we managed to get all the backbones in the right size. The first run we had a positive result only with the pT7 backbone, the second run we had only the K1F backbone and in the third run, after raising the Tm by 10 degrees, we had the rest of the plasmids backbones- T3, N4 and SP6.

Restriction, Ligation and Transformation

Figure 4: gel electrophoresis for the Colony PCR results of all the plasmids with phoA as an insert. Expected size: 1750 bp . The left lane is a standard 1 Kb ladder (NEB).  A- Four positive colonies for the pT7 backbone can be seen; control stands for PCR mix without a colony. B- Two positive colonies for the pN4 and pT3 backbones can be seen; the control is a ligation reaction without an insert. C- One positive colony for the pSp6 backbone can be seen; control is the PCR mix without a colony.

Figure 5: gel electrophoresis picture for the colony PCR results of all the plasmids with xyIE as an insert. Expected size: 1200 bp. The left lane is a standard 1 Kb ladder (NEB).  A- One positive colony for the pT7 backbone can be seen; control stands for PCR mix without a colony. B- At least one positive colony for the pT3, pN4, pK1F and pSP6 backbones can be seen; the pT3 and pSP6 controls stands for a ligation reaction without an insert, the "control- no colony" stands for the PCR mix without a colony.

We performed restriction with XhoI and XmaI restriction enzymes on both the plasmids and the inserts (phoA and xyIE) and ligated them together. In order to check whether the ligation was successful and we got ourselves new BioBricks we did a colony PCR with one primer for the insert and one for the plasmid.

phoA Colony PCR

As shown in Figure 4, we managed to ligate the phoA insert with most of our backbones, except for the K1F promoter backbone. This means that we got 4 new BioBricks :)

xyIE colony PCR

As shown in Figure 5, we managed to ligate the xyIE insert with all of our backbones. This means that we got 5 new BioBricks :)

Controls

Positive Controls

Figure 6:  gel electrophoresis for the PCR results of the positive control backbone (triplicate). Expected size: 2300 bp. The left lane is a standard 1 Kb ladder (NEB).

As mentioned before, after we made our new BioBricks we wanted to know whether the genes are expressed and the assays actually work. So we ligated our inserts- phoA and xyIE, to a backbone from the distribution kit (BBa_I13600), which contained the pTetO promoter along with an engineered CFP (eCFP) gene which we removed by PCR. We also added XhoI and XmaI restriction sites in both ends of the plasmid. 
As shown in Figure 6, the experiment yielded a positive result.

Figure 7: gel electrophoresis for the colony PCR results of the positive control plasmids with each insert. Expected size for phoA: 3750 bp, and for xyIE: 3250 bp. The left lane is a standard 1 Kb ladder (NEB).

Restriction, Ligation and Transformation

We performed restriction with XhoI and XmaI restriction enzymes on both the plasmids and the inserts (phoA and xyIE) and ligated them together. In order to check whether the ligation was successful and we got ourselves new BioBricks we did a colony PCR with one primer for the insert and one for the plasmid. uniform

As shown in Figure 7, we had positive colonies only for the xyIE and not for the phoA.

Figure 8: Presents a starter of the BL21 bacteria with the pTetO + xyIE plasmid. On the left is a tube with the addition of catechol, on the right is the one without an addition of catechol.

As shown in Figure 8, a distinct yellowish hue in the xyIE assay was detected after the substrate was added, which means our xyIE gene works as planned.

Negative Controls

1. The first negative control is a bacteria without the assay genes, which means the bacteria contains only the PET system. The results of the absorbance After induction with IPTG and performing the assays protocols are shown in Table 1:

   Table 1: Absorbance results for the first negative control, after blank reduction, for xyIE and phoA assays after induction with IPTG.

   	Absorbance
   xyIE assay(380 nm)	0.064
   phoA assay (420 nm)	0.000*

   * Because of the blank reduction the absorbance was negative, which is not a valid absorbance.

   As seen in the results, the absorbance acquired from the assays was very low. This indicates that there hasn't been enough enzyme in the bacteria that could have caused the yellow color.

2. The second negative control is a bacteria containing only the assay part- T7 promoter+ xyIE\phoA genes, without any RNAP. The results of the absorbance after performing the assays protocols are shown in Table 2:

   Table 2: Absorbance results for second negative control after blank reduction for xyIE and phoA.

   	Absorbance
   xyIE (380 nm)	1.200
   phoA (420 nm)	0.171

   As seen in the results, the absorbance yielded from the assays was low in comparison to the assay results from the pET experiment, [0.5 to 1.5] for the phoA and [10 to 60] for the xyIE. This indicates that the T7 promoter has a very low leakage level.

Characterization of the Assays

As mentioned before, the characterization will be done by adding different concentrations of the inducer, IPTG, while the saturation time and amount of substrate for the assay are identical.

Alkaline Phosphatase

The graph below presents the results of the experiment described. The absorbance at 420nm for each of the different concentrations of the inducer in a range of 0.5 μΜ to 1000 μΜ on a logarithmic scale. The absorbance was calculated via different dilutions of the samples: dilutions by 0.5, by 0.25, and by 0.1. The error bars represent the processing of the data collected. The line at the bottom of the graph represents the basal level according to the control result- no IPTG induction.

Figure 9: absorbance at 420nm against concentration of inducer

As can be seen, the graph shows a clear positive tendency- the higher the concentration the higher the absorption, as expected. Starting from a concentration of  40 μM and above, there are only small deviations from the absorption value of  1.2, probably due to the fact that saturation has been achieved.

xyIE

The graph below presents the results of the experiment described. The absorbance at 380nm for each of the different concentrations of the inducer in a range of 0.5 μM to 500 μM on a logarithmic scale. The absorbance was calculated via different dilutions of the samples: dilutions by 0.5, by 0.25, and by 0.1. The error bars represent the processing of the data collected. The line at the bottom of the graph represents the basal level according to the control result- no IPTG induction.

Figure 10: absorbance at 380nm against concentration of inducer.

As can be seen, the graph shows a small tendency to rise, but it's not clear enough. There are several explanations, one is that saturation has been achieved from lowest concentration measured, which shows both that the assay is highly sensitive and that a small amount of polymerase is enough to cause translation. The experiment should be repeated with either lower concentrations of IPTG or Catechol. The line at the bottom of the graph represents the basal level according to the control result- no IPTG induction

Conclusions and summary

• We have produced two separate assays for testing the presence of the RNA polymerases that is used for the induction of the phage.
• Since both of our assays show very low leakage the possibility of a getting a false positive result is not high.
• Since in both cases very small amounts of IPTS showed positive results, we can deduce that even a small amount of the RNA polymerase can activate the promoters. In terms of the Trojan Phage, that can create a situation that even if there are no activators present in the cell, the leakage of the polymerase promoters can create enough polymerases to translate the phage's proteins and cause lysis.

References

1. Temme K., et al. 2012. Modular control of multiple pathways using engineered orthogonal T7 polymerases. Nucleic Acids Research (advance access): 1-9.
2. Shin I., Kang C. 2003. Mutational analysis and structure of the phage SP6 promoter. Methods in Enzymology 370: 658-668.