Determing the plasmid copy number

The foundation of multi-plasmid framework is the ability to determine plasmid copy number (PCN) per cell. Our approach to count the PCN in the cell is built upon absolute quantitative PCR.

Why we chose a specific set of primers for PCN determination?

Commonly, gene used for PCN evaluations in qPCR is an ampicillin resistance gene (bla). In our case, the bla gene for plasmid number determination was not used, as the SynORI multi-plasmid framework employs a number of plasmids with different selection system gene circuit parts. Instead, the origin of replication was barcoded with distinct sets of primers (named qPCR) for different groups of origin copy number determination. This enables to determine the desired plasmid group copy number, when working with multi-plasmid systems.

Using cell lysates instead of extracted DNA

Since working with extracted genomic, plasmid or total DNA incorporates an error which depends on the extraction efficiency, we decided to work with specially prepared cell lysate suspensions, skipping the extraction step. Furthermore, dxs gene from chromosome was ligated into pUC19 plasmid for less complicated standard preparation.

Results

First of all, it was concluded that primers, designed for calculations, are appropriate for absolute quantitative PCR by measuring the efficiency of PCR amplification reaction. Both genes have almost ideal amplification efficiency and required no further optimization.

Figure 1. PCR amplification efficiency of genes used for plasmid copy number evaluations.

Amplified qPCR product were verified using agarose gel electrophoresis.

Figure 2. Verification of products amplified during qPCR.

Figure 3. Melting curves of amplified qPCR products used for PCN determination.

Next, standard curves were generated for dxs and qPCR plasmid gene copy number determination according to the protocol written by our team (click here?).

Figure 4. Standard curves generates for plasmid copy number determination for SynORI project.

The fit of the linear regression model was satisfactory; the coefficient of determination (R2) was more than 0.999 for all standard curves. As amplification efficiencies and generated standard curves were ideal for both genes, the curves and earlier mentioned protocol were used for all further PCN determination experiments.

Refference:

Plotka M, Wozniak M, Kaczorowski T. Quantification of Plasmid Copy Number with Single Colour Droplet Digital PCR. Doi H, ed. PLoS ONE. 2017;12(1):e0169846. doi:10.1371/journal.pone.0169846.

Anindyajati, Artarini AA, Riani C, Retnoningrum DS. Plasmid Copy Number Determination by Quantitative Polymerase Chain Reaction. Scientia Pharmaceutica. 2016;84(1):89-101. doi:10.3797/scipharm.ISP.2015.02.

Plasmid copy number control

Flexible copy number control is the core of our framework, which is based on re-engineered ColE1 origin of replication.

Base of SynORI framework - ColE1 replicon

ColE1 plasmid replicon is based on two antisense RNA molecules: RNA I and RNA II.

The transcript of RNA II forms a RNA-DNA duplex and acts as a primer for DNA polymerase and for that reason is often called a replication initiator.

During the transcription of RNA II, several different secondary structures can form. Part of these structures are susceptible to the binding of RNA I - a shorter antisense version of RNA II. The interaction between RNA I and RNA II begins upon the formation of kissing-loop pairs between their anti-complementary secondary structures. If the kissing complex persists, a 3’ end of RNA I starts forming a zipper-like duplex with a complementary region of a single strand RNA II. Because the primer cannot be formed anymore, this results in replication inhibition, which is why RNA I is often called the replication inhibitor.

The main reasons why we have chosen ColE1 as base for SynORI framework were:

• Its proficient biochemical and mathematical characterization;
• Its simplicity for consisting of only two regulatory RNA molecules;
• The favor of kissing-loop complex formation kinetics to predict plasmid group compatibility.

Picking the control type

It immediately becomes clear that in order to control the copy number of a plasmid, one could simply change the RNA I promoter. However, there is a reason why it has never been done before!

As mentioned above, RNA I and RNA II are two antisense molecules, so it is impossible to change one without altering the sequence of another molecule. RNA I promoter is located right on top of the RNA II secondary structures, which are not used for inhibition, but are crucial to form the RNA-DNA duplex for replication initiation.

Even if one managed to somehow substitute the RNA I promoter with another without disabling replication, it would still be inconvenient because this method would require a large pool of resources every time.

For that reason we have decided not to modify or replace the RNA I promoter inside the initial (laukinio tipo?) wild type origin of replication, but rather to disable it completely and place a copy of it next to RNA II.

Disabling the RNA I promoter

As already indicated, the main problem of inactivating the RNA I promoter is the necessity to take precautions in order keep the critically important secondary structures of RNA II intact.

We have first acquired a priority mutation list from the literature. The list analysed RNA polymerase binding affinity to -10 and -35 promoter region and its dependence on point mutations, with mutations causing the largest decrease in affinity being displayed on the top of the list.

Table 1. Priority mutation list of -10 and -35 promoter regions.

We compiled a simulative algorithm which compared every possible combination of -10, -35 mutations and then matched them to predicted RNA II secondary structures made by CoFold, a thermodynamics-based RNA secondary structure folding algorithm that took co-transcriptional folding into account. Afterwards we set a replicon mutants prioritizing:

• Mutants that had unchanged RNA II secondary structures.
• Mutants that were on the top of mutation priority list (meaning the lowest RNA polymerase affinity).

Sequences of the 5 best mutants after running simulation:

WT

ori2

We had ordered the selected ColE1 mutants from the IDT and later tested if we had successfully disabled the RNA I promoter.

It is difficult to distinguish when the promoter is fully disabled because first, there is no literature data describing replicons that are not negatively regulated at least to some extent, and second - plasmid systems hardly reach the equilibrium without negative control therefore every copy number calculation varies greatly. This is why we decided not to check for the highest copy number mutant, but rather to insert a wild type RNA I with its wild type promoter. By doing that we could see which replicons were most precisely mutated.

If the plasmid copy number (PCN) did not differ from wild type after the insertion of an RNA I gene next to the mutated replicon, it proved a complete disabling of the replicon. Contrary, if the copy number decreased, we could suspect that the replicon did not have a completely disabled RNA I and the sum of inhibition from both RNA I genes reduced the copy number to even lower values than in the wild type replicon.

First, we planned to calculate the copy number of our mutants that supposedly had their RNA I gene promoter disabled (mutants ORI 1, ORI 2, ORI 3, ORI 4, ORI 5). After that, we aimed to calculate the copy number of the corresponding mutated replicons, but with RNA I gene containing its wild type promoter cloned next to them.

After transformation, cells with ORI 5 plasmids did not grow successfully, which suggested a conclusion that this mutant had either severely damaged RNA II gene or increased expression of RNA I to the level of complete replication inhibition.

Since 4 other mutants had grown after the transformation, we incubated the cells overnight, purified the plasmids and cloned wild type RNA I with its wild type promoter next to each of the mutants. We then calculated the copy number of 8 samples: 4 ORI mutants and 4 ORI mutants with RNA I placed next to them.

Figure 1. Copy number calculations of the RNA I promoter elimination mutants. Two biological replicates were performed, with 2 technical qPCR replicates each time.

Firstly, ORI 1 mutant had a moderate increase in copy number (Figure 1). Yet, with RNA I next to the replicon, the copy number did not seem fall back to wild type levels. We hypothesize that the reason for this was the damage done to the RNA II gene. The damage resulted in mutant formed secondary structures no longer sufficiently interacting with inhibitory RNA I molecules.

ORI 3 did not seem to increase much in copy number. We did not consider it to be a good candidate as well, because we wanted our core synthetic ori to possess a range of copy numbers to choose from.

The third candidate, ORI 4, seemed to be a decent candidate because with cloned RNA I its copy number fell to near wild type levels, but it also did not prove to be good enough, because its maximum number of copies was too low.

ORI 2 mutant seemed like a perfect candidate. Its copy number increased from wild type X levels to Y +- Z. In addition, when RNA I gene was placed next to it, the copy number of the constructed plasmid fell to wild type levels. After these results we have decided to use this ORI 2 mutant as a core for our framework. We simply called it RNA II (Part:BBa_K2259000 ).

Tailoring the copy number control

Once the RNA I promoter was disabled in the ColE1 origin of replication, it could be moved to a different plasmid location and used as a separate unit. Also, RNA I promoter can be changed without having to worry about damaging the initiation of replication. RNA I and consequently the copy number of a certain plasmid can now be placed under virtually any signal pattern required.

We have discovered the sequence of wild type RNA I promoter by using PromoterHunter. and ordered a wild type RNA I gene from IDT without the promoter’s sequence. We have first cloned series of anderson promoters next to the RNA I gene and then placed this construct next to RNA II (RNA II-Anderson-RNA I).

Figure 2. RNA I and RNA II constructs, with RNA I constructs under different-strength Anderson (See anderson collection here http://parts.igem.org/Promoters/Catalog/Anderson ) promoters.

In theory (see "Modelling" for more details), lower-strength Anderson promoters should yield lower concentrations of RNA I, hence higher copy numbers of plasmids per cell. Our constitutive copy number device experiment results prove it to be true in practice as well. The stronger Anderson promoter is used, the less copy number per cell we get. With the strongest Anderson we get only 21+-6.84 plasmids per cell.

Worth to mention is that the closest to wild type ColE1 replicon is the 0.86 strength Anderson promoter, measured by copy number alone. (Part:BBa_K914003).

For this experiment we have built a rhamnose and RNA I construct (Part:BBa_K2259065) and then cloned this construct next to RNA II (Part:BBa_K2259091). We have used different percent of rhamnose in our media in order to see if this approach was possible and if so, to figure out the dependency between the plasmid copy number and rhamnose concentration.

Figure 3. RNA I and RNA II constructs, with RNA I gene being under the rhamnose promoter.

The first thing we noticed was that rhamnose promoter was very strong in terms of plasmid copy number reduction. It was also considerably leaky (promoter can be enabled even without any inducer). At zero induction there were approximately only 9 plasmids per cell and at 1 percent induction the number rose to approximately 1 plasmid per cell.

RNA I rhamnose-induced promoter seemed to be working well, with higher concentrations of inductor giving lower plasmid copy number.

We called it the SynORI copy number induction device.

So now when we can flexibly control the copy number of a plasmid, the only question is - what will come next?

References

Tomizawa J. Control of ColE1 plasmid replication: the process of binding of RNA I to the primer transcript. Cell. 1984 Oct;38(3):861-70.

Camps M. Modulation of ColE1-Like Plasmid Replication for Recombinant Gene Expression. Recent Patents on DNA & Gene Sequences. 2010 Oct; 4:58-73

Som T, Tomizawa J. Regulatory regions of ColE1 that are involved in determination of plasmid copy number. Proc Natl Acad Sci U S A. 1983 Jun; 80(11): 3232-3236.

Tomizawa J, Itoh T, Selzer G, Som T. Inhibition of ColE1 RNA primer formation by a plasmid-specified small RNA. Proc Natl Acad Sci U S A. 1981 Mar;78(3):1421-5.

Masukata H, Tomizawa J. Control of primer formation for ColE1 plasmid replication: Conformational change of the primer transcript. Cell. 1986 Jan; 44(1): 125-136.

Brenner M, Tomizawa J. Quantitation of ColE1-encoded replication elements. Biochemistry. 1991 Jan; 88:405-409.

Brewster R. C, Jones D. L, Phillips R. Tuning Promoter Strength through RNA Polymerase Binding Site Design in Escherichia coli. PLoS Comput Biol. 2012 Dec; 8(12): e1002811.

Multiple plasmid groups

Multi-plasmid framework would not be much without multiple plasmids. We have equipped our synthetic origin of replication with specific sequences to create unique plasmid groups.

PAs RNA I and RNA II interact mainly with the three stem loops that form kissing complexes, we have decided to use this feature to our advantage in order to engineer different plasmid groups by adding unique, group-specific sequences to RNA I and RNA II stem loops.

The specific sequences were acquired from Grabow et al., where they have screened a large number of different RNA kissing stem loop complex combinations. They have derived a table of different loop sequences that only bind with each other but do not have any cross interaction to the following loop sequences in the list.

The inactivation and transfer of RNA I gene away from RNA II allows us to use different sequences for RNA I and RNA II molecules that are not necessarily ideal complements of each other.

Since there are three stem loops responsible for RNA I-RNA II interaction for each of the plasmid group we have decided to:

1. Use two different unique sequences in the first two RNR I and RNR II stem loops, in order to maximize same group specificity.
2. Keep RNA II unchanged for the third loop but change it in the RNR I by adding either G/C mutations to RNA I (GC type RNA I) or making RNA I completely non-complement to RNA II (NC type RNA I).

According to literature (Tomizawa, 1984) RNA II secondary structures are very sensitive to any mutations in the third loop and has a high chance of ruining the replication initiation. To avoid this we did not want to alter the third loop of RNA II sequence. Just because we chose not to interfere with the third loop of RNA II, we could not leave the third loop of RNA I unchanged. If every group had a fully compatible third loop, the background cross-group inhibition would be too large and we would not be able to obtain independent plasmid groups.

We have designed 5 different RNA II genes corresponding to groups A, B, C, D and E and needed to make sure if they were working (Figure 2).

Figure 2. Table displaying RNA II of plasmid groups according to their loop sequences. Group A contains 1 kissing-loop from Figure 1 in both first and second loops, group B - h and a, group C - c and d, group D - l and e, group E - f and g, first and second loop respectively. All plasmid groups third loop corresponds to one found in the wild type.

Figure 3. Different plasmid groups can be co-maintained in a cell with a specific, pre-selected copy number. Copy number control principle is the same for every plasmid group, but both RNA I and RNA II molecules are only specific to their own.

These different plasmid groups can then be co-maintained in cell with a specific, pre-selected copy number. Copy number control principle is the same for every group, but each group is only specific to its own group.

Results

Choosing the suitable pairs

The first step in experimentally choosing the suitable RNA I-RNA II pairs was done by employing a system that was able to measure the trans-acting effect of each RNA I type of group A has on the RNA II group A (BBa_K2259000) (RNA I types: WT (BBa_K2259005), NC (BBa_K2259024) and GC (BBa_K2259006)). By co-transforming base vector 2.0 (BBa_K2259081) having an A group RNA II together with pSB4A5 plasmid, which is constantly expressing one the mentioned RNA I. The plasmid copy number per cell was determined for each co-transformation using the absolute quantification qPCR.

Figure 4. The trans-acting effect each RNA I type has on A group RNA II plasmid copy number

As seen from the results, the GC type RNA I has the lowest tendency to cross interact with the RNA 2 initiator of replication from group A, as the copy number of the base vector 2.0 drops by the smallest amount. RNA I of NC type has the biggest cross-interaction level as it inhibits the plasmid replication the most.

Group analysis of RNA I - RNA II type GC.

After the degree of interactions have been established amid A groups of all types RNA II-RNA I, GC stem loop structures have been chosen for further group interaction assessment. First of all, we turned to determining the copy number each group of RNA II (A - BBa_K2259000, B - BBa_K2259001, C - BBa_K2259002, D - BBa_K2259003 and E - BBa_K2259004) establishes in the cell with and without its corresponding RNA I constantly expressed by the Anderson promoter in the same vector.

Figure 5. Plasmid copy number each group of RNA II establishes in the cell with and without its corresponding, constantly expressed, RNA I.

Seeing that group E and C measurements were off, we concluded that RNA II did not fold into correct secondary structure and, as a result of that, it did not work as a primer formation unit. We chose to work with the remaining three groups as the plasmid systems produces stable plasmid copy results. B group RNA II seems to have enhanced primer formation abilities, as the plasmid copy number skyrockets. Every RNA II construct is sensitive to its RNA I, as the plasmid replication is inhibited by constantly expressed RNA I transcript.

Cross-interactions of type GC

Type GC stem loop cross-interactions were assessed by constantly expressing RNA I transcript from pSB4A5. The experiments were rather tricky, as the pSB4A5 plasmid seemed to be easily lost when co-transformed with our base vector 2.0. Nevertheless, we were able to measure the cross interactions between the groups displayed in Figure 6.

Figure 6. Cross-interaction assessment of different groups of RNA I-RNA II.

As seen from Figure 6, there is no interaction between these groups: A and C; A and B. The inhibition has been identificated between B and D; A and D groups. Based on the following results, 2 types of plasmid groups were chosen for 2 co-transformation plasmid copy number assay: A with B and A with D groups.

2 plasmids co-transformation copy number assay

Group A RNA I-RNA II has been co-transformed with group B RNA I-RNA II and vectors with and without global copy number control devices (BBa_K2259052, BBa_K2259053, BBa_K2259075).

Figure 7. Plasmid copy determination of two different plasmids in the same cell.

Based on the results from Figure 7, it can be concluded that RNA II A and RNA II B act as different replicon and does not inhibit the replication of each other. By introducing the global copy number device, plasmid copy number decreased equally of both plasmids.

References

Grabow WW, Zakrevsky P, Afonin KA, Chworos A, Shapiro BA, Jaeger L. Self-assembling RNA nanorings based on RNAI/II inverse kissing complexes. Nano Lett. 2011;11(2):878–87.

Singh A, Sethaphong L, Yingling Y. Interactions of Cations with RNA Loop-Loop Complexes. Biophysical Journa.l 2011 Aug; 101:727–735

Gregorian R., S, Donald M. CrothersDeterminants of RNA Hairpin Loop–Loop Complex Stability. J. Mol. Biol. 1995; 248:968–984.

Eguchit Y, Tomizawa J. Complexes Formed by Complementary RNA Stem-loops Their Formations, Structures and Interaction with ColEl Rom Protein. J. Mol. Biol. 1991. 220:831-842.

Lee A, Crothers D. The solution structure of an RNA loop–loop complex: the ColE1 inverted loop sequence. Structure. 1998 Aug; 6:993–1005.