Introduction

Plasmids are DNA entities that replicate independently from the chromosome. They are used as vectors for the production of recombinant proteins or genes for research and commercial purposes. ColE1-like plasmids are the most popular ones because they are maintained at high enough copy number in the cells. This increases the yield of DNA and/or helps raise the level of expression of the desired recombinant product.

The product of a Gibson Assembly is a fully ligated double-stranded DNA molecule of the plasmid and the target gene. It’s crucial to know if the plasmid has a high or low copy number before starting the experiment. PCN information is useful for effective troubleshooting. Plasmid copy number affects the efficiency of Gibson assembly and ultimately transformation which is mainly the use of exogenous DNA to produce proteins encoded in the engineered DNA. Efficiency of assembly decreases as the number or length of fragments increases. Controlling plasmid copy number helps improve the yield of plasmid DNA and protein for research and biotechnological applications. In order to save time and resources, it’s important to know the plasmid copy number to predict yields of assembly reactions and even when they will not work.

An advantage of a high plasmid copy number is the greater stability of the plasmid when random partitioning (i.e., partitioning of plasmids into daughter cells) occurs at cell division. As much as high copy number brings stability at the cell division level, there is a concern about the level of toxic products produced. In our project we utilize a kill switch as a safety measure. If we consider as low plasmid numbers as possible, we can reduce the levels of toxic products in the bacteria which is another layer of safety for the implementation of our biosensor.

Mutations were introduced into pSB1C3 origin of replication. The pSB1C3 plasmid is a high copy number plasmid that has chloramphenicol resistance. It has terminators around the multiple cloning site (MCS) that help to prevent transcription within the MCS from reading out into the vector. Some of the mutations increase the PCN and others reduce it. Many steps have been taken to prove that the mutations indeed altered the plasmid copy number of the wild type.

In the validation process, qPCR and gel electrophoresis have been used to measure the plasmid and genomic copy numbers. Multiple qPCRs and gels have been run with the mutants and wild type. The results so far show that there has been a successful alteration to an existing wild type to produce both the loss of function and gain of the function gene. All processes have been repeated to further justify the results obtained by far. It's still a work in progress.

Sample	CT Number
Camp -13(DNAe)	33.01
Camp -13(ori SeqQ F&R-b)	27.47
Camp+8 (DNAe)	20.52
Camp+8 (ori SeqQ F&R-b)	14.81
WT (DNAe)	18.03
WT(ori SeqQ F&R-b)	16.47
Camp -11(DNAe)	23.44
Camp -11(ori SeqQ F&R-b)	28.33
Camp +8 -11(DNAe)	32.09
Camp +8 -11 (ori SeqQ F&R-b)	13.97
A715G (DNAe)	18.85
A715G (ori SeqQ F&R-b)	24.52
Camp +1 (DNAe)	No CT
Camp +1(ori SeqQ F&R-b)	13.43
Control (DNAe)	No CT
Control (ori SeqQ F&R-b)	No CT

Figure 1: qPCR amplification plots of 6 mutants and a wildtype

Figure 1 above shows how the qPCR system detects the cycles through fluorescence. The system sets an automatic threshold above which the CT numbers will be measured. CT value is the number of cycles required for the fluorescent signal to exceed background levels. LUNA dye is one of the components of the PCR which serves as the source of the fluorescence. Any flat line means that sample had no starting DNA to multiply. Each cycle doubles the amount of DNA present in the sample. A single difference in CT numbers between two samples means there was two times more DNA in one sample than the other.

Plasmid copy number = 2^{(CT of WT - CT of mutant)}

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The CT values in the data above suggest changes in plasmid copy number in the wild type as found in the mutants. After comparisons and calculations, it was found that the mutants have the following plasmid copy numbers relative to the wild type.

Camp +8 has two times more plasmid

Camp +1 has eight times more plasmids

A715G has 1/256 times plasmids

Camp +8 -11 has about 5 times more plasmid

Camp -11 has about 1/4096 times more plasmid

Camp -13 has about 1/2048 times more plasmid

As expected, all the negative mutations lowered the plasmid copy number and all the positive mutations increased the copy number. To further justify the conclusions from the qPCR, gel electrophoresis was also used to measure the plasmid copy number of the mutations. The data is also presented below. This data doesn't exactly correlate with the qPCR but it's good enough to support the conclusions that indeed the plasmid copy number of the wild type has been altered with the introduction of different mutations to the origin of replication.

In the negative mutations, the loss of function genes are produced and the gain of function in the positive. The mutations cause loss or gain function by doing the following:

Camp + 8 causes runaway replication, -11 prevents R-loop formation and destabilizes, camps +1 increases P2 expression,and stabilizes secondary structure in promoter, camps +8/-11 causes both plasmid replication and prevents R-loop formation, A715G causes more kissing complexes that lower replication and lastly camps -13 causes defects in RNAseH-dependents and independent mode and conformational changes.

Gel Electrophoresis Data

Serial dilutions of pure DNA of all mutants and a wild type were run on different gels to separate the genomic and plasmid DNA for the analysis. The gel below represents the approach used to determine the plasmid copy number of all samples in the analysis. The top band represents the genomic DNA and the lower bands the plasmid DNA. The bands show exactly what was predicted. The concentrations decrease with increasing dilutions and so the bands are expected to decrease in intensity from the side of the ladder. Plasmid DNA is smaller than genomic DNA so the base pairs must be smaller than the genomic DNA which is what the gel shows.

Figure 2. Camp +1 gel electrophoresis

Table 2. Camp +1 gel bands signals

Image Name	Channel	Name	Signal	Total	Area	Background	Type
GA_3	600	3kbp band	23.5	32.2	651	0.0134	Signal
GA_3	600	1 top	4.13	11.1	741	0.00935	Signal
GA_3	600	2 top	2.54	8.27	665	0.00861	Signal
GA_3	600	3 top	1.41	7.76	703	0.00903	Signal
GA_3	600	4 top	0.907	6.44	646	0.00856	Signal
GA_3	600	5 top	0.328	4.17	456	0.00842	Signal
GA_3	600	6 top	0.190	6.34	731	0.00842	Signal

The genome size is about 4,600,000 bp and the plasmid size about 3000 bp. It is expected that the mutations cause either an increase or decrease in the plasmid size which is what qPCR has already proven in the data presented above. The plasmid copy numbers(PCNs) were calculated with the formula below.

Plasmid signal/plasmid size = PCN x Genomic signal/Genomic size

The wild type (pSB1C3) has a copy number range between 100 and 300 per cell. Some of the gel signals were below detection but it was assumed that those could be half the preceding signal because they were all serial dilutions. The calculated copy numbers for the mutations based on the gel results are as follows:

Camp +1 = 307

Camp +8 = 381

Camp +8 -11 = 163

Camp -13 = 133

Camp -11 = 194

A715G = 102

While the exact plasmid copy numbers for the mutations vary with different methods, they give a similar trend. The positive mutations result in increased copy numbers while the negative reduce them just as predicted.

The purpose of the kill switch is to ensure a safe use of the biosensor. Plasmid copy number determines the dosage of gene available and accessible for any form of expression. A lot of plasmids lead to high product production so if those products are environmentally unfriendly that creates a new problem in an attempt to find a solution to existing problems. Engineered bacteria could be controlled to produce minimal products if they can potentially be harmful. One way of doing that is to lower the plasmid copy number. The PCN could be altered to a level where it's just enough to allow the bacteria to function but minimal enough to avoid the effect of its products. That's another level of safety possible to incorporate in the implementation. When the copy number is high, the cell reproduces and creates products rapidly so if mutations can reduce copy number then the rate of production can be controlled.

LAC I gene in the bacteria genome codes for products that repress the expression of red fluorescent protein (RFP). The copy number of the RFP plasmid is important to determine the level at which RFP can be expressed. Due to the mutated plasmid containing the genes for red fluorescent protein (RFP) controlled by a Lac promoter, if there are too many copies of the plasmid within the bacteria, then LacI cannot inhibit all the plasmids. Plus mutations are expected to increase the copy number and therefore make it difficult for LAC I to inhibit RFP expression. Conversely, minus mutations will make it easier for LAC I to inhibit RDP because they reduce the copy number.

The image on the right shows how LAC I inhibits RFP and the colors they produce depending on how many plasmids present. The positive mutations are brighter and the negative ones are faint. The brighter the streaks the more plasmid present and vice versa. Listing below is what 1 through 8 represents:

1 = WT, 2 = A715G Mutation, 3 = +1 mutation, 4 = +8 mutation, 5 = -11 mutation, 6 = +8/-11 mutation, 7 = -13 mutation, 8.= double inverter

Figure 3: A plate of bacteria streaks