Team:Newcastle/Measurement

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Measurement

Background

While the InterLab study is an effective way of gathering large sets of data surrounding the parts and protocols, it does not consider the variability between data sets. As there are many factors which cause variation in microbial protein expression and productivity, some factors may be more favoured than others, leading to an overrepresentation of these in the metadata. Bio-design Automation (BDA), an emerging field focused on computer-aided design, engineering principles, and automated manufacturing of biological systems (Appleton, 2017), is the obvious solution to this problem as it will allow faster and better high throughput analysis of large data sets – giving BDA the potential to have a significant positive effect on the progression of synthetic biology. However, the complex nature of biological systems can make accurate characterisation of parts quite difficult, and since models driven by BDA require such accurately characterised parts, the scope of BDA in synthetic biology is currently lower than we would have hoped.

We aimed to solve this problem by implementing our own internal standard (IS) and fluorescent reporter. The IS, a medium strength, constitutively expressed RFP construct, was cloned into each test device pSB1C3 backbone. The theory is that since each test device vector contains the same IS, their relative fluorescence regarding RFP should be similar or the same. The IS will help us determine if the characterisation of the devices is valid and will aid in identifying the sources of variation – since the protocol and equipment will be identical, variation will be expected to come from other sources which are often unaccounted for.

Moreover, we aimed to reduce stochasticity in part characterisation by replacing the GFPmut3b reporter with a brighter, more photostable fluorescent protein named mNeonGreen (Shaner et al. 2013; Balleza et al. 2018). The mNeonGreen protein is widely used in the imaging of cellular components due to it having a fluorescence 3-5 times that of GFP. However, there is little indication in the literature that it has been used as a reporter for the characterisation of circuits. Replacing mut3GFP with mNeonGreen will allow the investigation of whether the fast folding capabilities coupled with its brightness and higher photostability will yield a lower spread of fluorescence values regarding the original GFPmut3b, making it a better tool for part characterisation.

Internal Standard (IS) and mNeonGreen Design, Assembly & Analysis procedure.

Purification of pSB1C3 from transformed E. coli DH5α

All 6 test device plasmids and the controls were purified from the previously transformed E. coli DH5-alpha cells using the Qiaprep® Miniprep Kit & protocol by Qiagen®. A Q5 polymerase PCR protocol (NEB) was used via the VFR and VF2 primers to determine presence of the plasmid.

Design of IS

The RFP IS construct was designed using Benchling (Figure X). The parts used for building the RFP construct were Anderson promoter BBa_J23108, RBS BBa_0032, the RFP gene and double terminator BBa_B0015. Gibson ends were also designed for cloning into pSB1C3 using the NEBuilder DNA assembly tool and the gBlock was synthesised by IDT. The promoter has a measured strength of 0.51 relative to BBa_J23100.

Design of mNeonGreen

The mNeonGreen construct (Figure X) was designed for use as an alternate fluorescent reporter for each test device - replacing GFPmut3b. The mNeonGreen sequence was codon optimised for expression in E. coli DH5α using Benchling and the Gibson ends were designed using NEBuilder for cloning into pSB1C3. The subsequent design was synthesised by IDT.

Cloning of gBlocks into pSB1C3

Plasmid concentration for each mini-prepped test device was determined using a Qubit fluorometer and diluted to 0.5 ng/µl. The diluted pSB1C3 vectors were linearised using a 2 step PCR system following a Q5 Polymerase protocol (NEB). This protocol utilised forward and reverse primers with Tm values of 72°C. For the IS, test device pSB1C3 were linearised via 2-step PCR at a non-coding region between the ORI and chloramphenicol resistance gene, a region deemed suitable due to its distance from the multiple cloning site. For mNeonGreen Six reverse primers complimentary to each of the test device RBS and their varying promoter regions, and 1 a single forward primer to bind at the beginning of the terminator, were utilised in 2-step PCR to linearise the respective pSB1C3 vectors - removing GFPmut3b. The amplified DNA was then digested with DpnI, heat treated to inactivate the enzyme and assembled via Gibson Assembly using the NEBuilder HiFi DNA Assembly Kit. Following their protocol, a 2-fragment reaction with 0.5 pmol of DNA in a 2:1 insert to vector ratio was done and transformants were plated onto agar plates with the appropriate antibiotic (LB+cam). Following growth of colonies, plasmid DNA was purified and sequenced to verify presence of the genes.

IS Analysis

Analysis of the IS involved comparing the original InterLab test device plasmids against the new IS plasmids. Microtiter plate rows A-D represented the RFP containing E. coli and rows E-H represented the original test device containing E. coli. Column 9 wells A-H contained an LB+cam blank. The microtiter plate was incubated for 24 hours in the plate reader with Abs600, fluorescence (GFP): Excitation 485, Emission 420 and fluorescence (RFP): Excitation 588, emission 635 measured every 30 minutes following a short shake at 420 rpm at a low shake diameter.

Over the 24-hour period the original test devices performed as expected, following the same pattern as the original InterLab study. IS fluorescein/OD data revealed a large difference when compared to the original. Firstly, devices 4 & 1, the groups with the strongest promoters and the most fluorescent, in this case, were not near as fluorescent as their original counterparts – with devices 2 and the positive control yielding the highest fluorescein/OD. Moreover, it is noticeable that all test groups, over a 6-hour period, are much lower than their original counterparts. This points toward competition between the RFP and GFP for transcription and translation mechanisms. However, it is after 6 hours where there is a significant change – where the original test devices decrease in their fluorescence, the internal standard devices consistently increase through to the 24-hour mark.

The fluorescence/OD data regarding the RFP of the IS in each vector is shown in figure X. Interestingly, test devices 1 & 4 showed no fluorescence regarding RFP. The fluorescence/OD values for the remaining test devices showed a consistent positive increase over the course of the 24 hours. The results however were also relatively variable between groups despite being under the control of the same promoter. The positive control had the highest fluorescence/OD value, reaching a peak at 0.107, and was followed by devices 6, 3 and 2, with fluorescence/OD values of 0.064, 0.048 and 0.038 respectively.

mNeonGreen Analysis

Three further InterLab studies were carried out for mNeonGreen expressing E. coli DH5α and those containing the original test devices, using the same conditions as the original study (2 single colonies chosen from each of the transformants for analysis). The fluorescein/OD of the mNeonGreen study was compared to the original InterLab study.

Fluorescein/OD data is represented in figure X. The figure shows that the fluorescence patterns in mNeonGreen are similar to those of the original test devices, with 4 & 1 being the strongest promoters and 3 being the weakest. At 6 hours, mNeonGreen is seen to exhibit a much higher fluorescence when compared to GFPmut3b in each of the devices. Moreover, there is an observed difference in the spread of data between the two reporters. At 0 hours, the spread of the data is large and variable between the 2 colonies. However, hour 6 values for both colonies show that the spread of fluorescein/OD for mNeonGreen devices are lower in each test device group.

Conclusions

Both the IS and mNeonGreen experiments were successful in revealing that part characterisation can be improved by implementing these new aspects. Inclusion of the IS improved GFP expression consistency. Moreover, variability of the IS signal was seen across plasmids that differed in only test device promoter sequence. The stronger devices lacked RFP fluorescence and each fluorescing device bore its own unique fluorescence curve relative to RFP. The data generated using this approach suggests that - with just two reporter constructs on the plasmid - there is competition for cellular resources between the IS and test device and as a result, past measurement and conclusions regarding the strength of strong reporters should be treated with caution. For the future, the IS will prevent the need for such caution, as it reveals variation between devices and may be an incredibly useful tool for future part characterisation. Substituting GFPmut3b with mNeonGreen revealed that mNeonGreen may be a more useful reporter protein for the characterisation of genetic circuits due to its higher fluorescence and lower stochasticity. Both the internal standard and mNeonGreen reporter, if coupled together, have the potential to become an essential characterisation tool, enabling the advancement of synthetic biology by effectively characterising parts which can be utilised in BDA to create novel genetic circuits.