Engineering

On this part of our wiki, we would like to elaborate how we implemented the The Engineering Cycle in our project to design a protocol to grow S.elongatus optimally and overexpressing genes producing alkanes.

Introduction

Cyanobacteria are not used commonly in biosynthetic research, because of their long duplication time and multiple genome copies [1] . Therefore, in order to utilize cyanobacteria for our research, we would have to first design a protocol for the specific strain to have optimal growth, which is essential to have a certain degree of efficiency. As one of the challenges we encountered in this project cycle was that we do not have access to a microbiological lab that would have been more equipped to host our project, as a part of our design (in the engineering cycle), we aim to create a protocol for the growth of S.elongatus UTEX2973-T, the fastest cyanobacteria with a doubling time of 2.1h at the best growing conditions and is also natural competent [2][3], to be grown in a general lab that is not equipped to grow such bacteria. For instance, an incubator with lights installed.

Generally, to culture bacteria is not a very complicated task. They can be cultured either in liquid medium or on solid medium (i.e agar plates). So we designed, built and tested two protocols for each growth medium that should be executable in most labs.

In order to design such a protocol, all factors affecting cyanobacterial growth, excluding the medium, would need to be taken in consideration.

Design & Build 1

First, the most prominent property of cyanobacteria is its ability to do photosynthesis, thus light would be one of the most important growth factors. For this reason, the process of light measurement needs to be standardized. Normally, in lab settings the Photosynthetic Photon Flux Density (PPFD) in umol/s/m2 would be measured for when dealing with photosynthesis processes. The appropriate tool is however not always accessible in every lab. A more accessible device that has lux or lumens measurement is called Luxmeter, which can be bought easily for 20-50 € from most online retailers like Amazon. Since with a Luxmeter, light is measured with a different unit, we find the below mentioned online calculator very helpful. This might also be helpful for future iGEM teams.

“https://www.waveformlighting.com/horticulture/convert-lux-to-ppfd-online-calculator”

With this approach, a standardized protocol for anyone that has limited or no access to certain equipment could be built.

Test & Learn 1

Like any other bacteria, our cyanobacteria, S. elongatus, has its own requirements or optimal growth temperature. So our first approach (design) is to try to compromise one of the factors. We tested this approach by growing a batch in the incubator without lights and another at room temperature with lights. This was quickly proven to be very unsuccessful. It took more than 48 h to see any signs of growth after inoculation. In our next design, we knew better than to compromise any factors.

Design & Build 2

Since we could not install any lights in the incubator, we had to find another heat source, so that we could keep our samples out of closed spaces. Since the lab bench we were lended had a PCR tube Heater/Mixer Block lying around. We thought we could use it as our heat source.

Another factor that needed to be taken into consideration was carbon dioxide supply. Unfortunately, we didn’t have access to carbon dioxide tank supply, so we could easily supply our cultures with carbon dioxide. One of our first ideas was to cover medium with parafilm instead of loose caps to isolate the system because it is transparent and shadowing will be avoided. Furthermore, according to the Datasheet [4], Parafilm is gas permeable and its CO2 permeability is greater than O2.

Test & Learn 2

To test our hypothesis, we prepared three different falcon tubes of inoculated growth medium, two of which were covered with Parafilm and one was closed loosely with its cap. All three cultures were to be grown at 39° C under 200 umol/s/m2 light source from a desk lamp. This hypothesis was proven to be false, since after three days, there was only growth observed in the one tube closed loosely with its cap. This means, even though the parafilm’s permeability of CO2 was greater than that of O2, it was still not enough for the cyanobacteria.

From this attempt as well, we learned that even though the heat source significantly helped. The heat source might not heat the tube evenly throughout, since falcon tubes were not made for the Eppendorf Heater/Mixer Block. Hence, relying on atmospheric carbon dioxide supply was not an issue, but temperature and light supply should be improved.

On the other hand, our bacteria culture grown on solid medium had no significant issues. It grew at a good rate, on the make-shift heating system, under an old desk lamp while only relying on atmospheric carbon dioxide.

Design & Build 3

Luckily enough, we found an incubator with a glass door in the storage room next door to our lab space. After we got permission to use it, we planned to install LED lights in the incubator. This should significantly improve the light and heat source problem.

Test & Learn 3

As we tried to install the lights in the incubator, we realized that the moisture and heat inside the incubator could damage the circuit of the LED lights. Furthermore, connecting the cables was problematic since there were no power sockets inside the incubator. So we decided to just stick the LED lights on to the glass door with some tape and connect it to the nearest power socket.

We learnt that this approach works very well and by this approach we could grow our bacteria in liquid medium just as fast as we could grow them on the agar plates on our makeshift heating system. Also worth mentioning, glass test tubes with very loose metal caps were most likely a better container for heating, since glass is a better heat conductor than plastic. The metal caps are also very loose, which would allow better carbon dioxide exchange than normal screw caps.

Results

In order to compare the growth of bacteria, grown in liquid medium, OD measurements were taken before and after incubation, at 750 nm (chloroplast extinction coefficient. Due to limited access to the lab, we were only able to check on growth every three days.

Design – Build – Test – Learn; Alkane Production

Overexpression through exogenous plasmids

For the biofuel production, we were planning to improve the NADPH production and increase the AAR/ADO system using a bacterial model that was already established in e.coli [5] using the lambda red recombination system, when knocking out genes.

https://www.neb.com/products/n3033-pbr322-vector#Product%20Information

The over-expression of 3 different genes (zwf, AAR and ADO) was planned by introducing two low copy (pBR322) plasmids.

One of which should carry zwf. Zwf gene may be inserted into the Ampicillin resistance gene sequence by restriction digestion at BsaI and PstI sites.Host cells could then be transformed and afterwards screened by testing its tetracycline resistance. To increase production of plasmid copies, E.coli dh5alpha is known to have high transformation efficiency. When dealing with cyanobacteria however, modifying the plasmids to be methylated could increase the efficacy of plasmid uptake. [6]

In a second pBR322, we may introduce AAR and ADO together connected with a non-transcriptional-spacer between the SphI and SalI restriction sites. However, since this would interrupt the Tetracycline resistance gene instead of the Ampicillin, after transformation, transformed cells should be screened for its Ampicillin resistance.

Transformation in S.elongatus UTEX 2973-t could be performed according to the standardized protocols, in case of low number of transformants a dark incubation could be performed in order to improve it [7].

Genes Knockout; inhibition of irrelevant genes

Most of the desired KO are Pathway holes, when a certain reaction in a cell induced by an enzyme does not have an associated gene. In simpler terms, the origin of the protein is not characterized yet. In an attempt to solve this issue, we tried to search for each genes’ homologous by constructing a primer while taking the homologous gene sequence into account.

One possible problem is that the primer won’t stick to the genome, yielding no PCR reaction. In this case, another approach would be using protein inhibitors.[8]

After doing Blast, a hypothetical protein shares great similarity with gene homologs from PTA, showing a possible candidate.

EDD seems to be absent in cyanobacteria. EDD is an evolutionary gene related to ilvd which is present in cyanobacteria that produces Phosphogluconate dehydratase [9]. But the Knockout of this specific gene is not possible, since this is apparently an essential gene in cyanobacteria. Absence of this specific gene, would impair the growth of the cyanobacteria. To approach this, we propose to increase GND’s expression (a gene that produces the enzyme), in order to reroute as much carbon from EDD/ilvd.

[10] This GND overexpression will increase the CO2 levels in the carboxysome enabling a better yield of alkane due to more glucose production via the CBB cycle.

Some possible problems in these gene knockouts is the presence of multiple genome copies which could be avoided by routine PCR-screening of the desired gene after each knockout.

Once the model is assembled, an assay to measure the alkane improvement could be made. Alkane yields could be compared between modified strains to wild type strains. This could be done by growing them to the same optical density of 1.0, measured at 750 nm. After harvesting produced alkanes, gas chromatography would be an option for quantitative analysis of the produced alkanes.