Difference between revisions of "Team:NRP-UEA-Norwich/Results/Prebiotic"

To achieve the cloning, we digested the various DNA components (GlgX or GlgB sequence, pSB1C3 and IPTG-inducible promoter) according to the restriction digest protocol with the relevant restriction enzymes to create compatible ends. The digestions were then run on an agarose gel and gel extracted to yield the digested DNA components. These were ligated and transformed into competent E. coli DH5α cells. A colony PCR was undertaken and liquid cultures of colonies with the correctly-sized inserts were prepared. The plasmids were purified by plasmid prep and sent for sequencing. After sequencing a mutation in the terminator codon of the GlgX sequence was discovered and this was subsequently corrected with mutagenesis PCR to give the correct sequence.

RESULTS:

The resulting plasmids were GlgX with IPTG-inducible promoter, GlgX without IPTG-inducible promoter, GlgB with IPTG-inducible promoter, and GlgB without IPTG-inducible promoter.

Figure 1: Plasmid diagrams of pSB1C3 containing an IPTG-inducible promoter (lacI), RBS and GlgX (left, BBa_K1618026) and pSB1C3 containing only the GlgX genetic sequence (right, BBa_K1618025).

Figure 2: Agarose gel electrophoresis image after mutagenesis PCR. Colony PCR gel? The GlgX insert is 2019 base pairs long and comparing the bands on the gel to the 2-log ladder, the GlgX bands reside just above the 2000 marker, therefore suggesting GlgX as the insert. Genetic sequencing confirmed the GlgX genetic sequence in the pSB1C3 vector was correct. Lane key or label?

Figure 3: Plasmid diagram of pSB1C3 containing the IPTG-inducible promoter (lacI) RBS and GlgB (left, BBa_K1618022) and pSB1C3 containing only the GlgB genetic sequence (right, BBa_K1618000).

Figure 4: Agarose gel electrophoresis image for correct GlgB cloning. The GlgB insert is 2231 base pairs long and comparing the bands on the gel to the 2-log ladder, the GlgB bands reside just above the 2000 marker, therefore suggesting GlgB as the insert. Genetic sequencing confirmed the GlgB genetic sequence in the pSB1C3 vector was correct.

Determining if expression of glycogen branching and debranching enzymes affect E. coli glycogen content

AIM:

To analyse the effect of GlgX (glycogen debranching enzyme) and GlgB (glycogen branching enzyme) expression on glycogen content in E. coli. We initially set out to investigate by looking at the staining of whole-cell extracts with Lugol’s solution, a method which a previous iGEM team had used. The iodine in the Lugol’s solution intercalates in the glucan chains to give a coloured complex. Comparison between the colours observed can give an indication of the amount and branching structure of the glycogen within the sample₁.

METHOD:

Competent E. coli were transformed with GlgX and GlgB under the control of an inducible promoter and firstly grown in 10 mL LB media overnight at 37 °C with shaking. Each culture was used to inoculate 2 x 10 mL of fresh media, grown to an OD of approximately 0.6 and then IPTG was added to one of each duplicate culture and the cultures continued to grow, with samples taken after 1 hour, 3 hours and overnight. At each timepoint a 1 mL extract was taken, spun down to pellet the cells and this pellet was re-suspended in Lugol solution, similar to a method previously been used by the Edinburgh 2008 iGEM team.

The expected colour upon Lugol addition to glycogen is a dark brown colour, however, as shown in Figures 1-5, no sample has turned this colour with the majority of the colour seen identical to the orange/yellow of Lugol itself. We therefore repeated this method with nitrogen-limited, carbon rich minimal media M9 minimal media, which had previously been shown to lead to the accumulation of bacterial glycogen₂.

Cells grew very slowly under these conditions and only reached an OD of approximately 0.6 after 24 hours.

RESULTS:

Figures 1 -5 show images of pelleted bacterial cells from LB media, resuspended in Lugol’s solution, and in every case it was not different to Lugol solution alone. 18 hours after induction The results from high carbon:nitrogen media are shown in Figure 5, in which every sample has a darker, browner colour than Lugol itself indicating a higher glycogen presence compared to LB media. Unfortunately solid particulates remaining in the samples precluded quantifying the results by measuring the absorbance with a spectrophotometer. Whilst there was clearly more glycogen in these samples, it was not possible to distinguish colour differences between the branching and debranching enzymes or between the IPTG-induced and non-induced samples. Therefore it was decided to extract the glycogen from the bacterial cells to analyse it directly.

Figure 1: The pelleted bacterial cells from 1 mL overnight LB cultures with no addition of IPTG re-suspended in 100 uL Lugol’s solution

Figure 2: The pelleted bacterial cells from 1 mL of LB culture an hour after IPTG-addition re-suspended in 100 uL Lugol’s solution

Figure 3: The pelleted bacterial cells from 1 mL of LB culture 3 hours after IPTG-addition re-suspended in 100 uL Lugol’s solution

Figure 4: The pelleted bacterial cells from 1 mL of LB culture 18 hours after IPTG-addition, re-suspended in 100 uL Lugol’s solution. The lighter colours observed are due to solid particulates in the media.

Figure 5: Pelleted bacterial cells of the branching enzymes grown overnight in M9 minimal media with or without IPTG, and resuspended in Lugol’s solution.

References

1. Meléndez-Hevia E., Waddell T.G., and Shelton E.D., 1993, Optimization of molecular design in the evolution of metabolism: The glycogen molecule , Biochem Journal, 295, p. 477–83

2. Bajka B.H., Clarke J.M., Topping D.L., Cobiac L., Abeywardena M.Y., and Patten G., 2010, Butyrylated starch increases large bowel butyrate levels and lowers colonic smooth muscle contractility in rats , Nutrition Research, 30, p. 427–34

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Making constructs to express putative acyltransferases in plant chloroplasts

AIM:

The aim of our prebiotic is to produce acylated/butrylated starch in plants. Methods to chemically acylate starch purified from plants already exist, but as they use strong chemicals and require heating they are not environmentally friendly. Using various acyltransferases, we hope to acylate starch in plants. We’ll be using a model plant, Nicotinia benthamiana, for initial tests because we can get results within a few days. Later on we’d aim to make transgenic plants in a species that makes a lot of starch such as maize (corn), potatoes or wheat.

Confirming the sub-cellular localisation of acyltransferases in plant chloroplasts

AIM:

To Determine if our synthetic chloroplast transit peptide would direct the putative acyl trasnferases to the plant chloroplast. The aim of this experiment is to confirm that our constructs localise the acyl transferase to the plant chloroplast. In order for the enzymes that we have chosen to acylate starch, the enzymes need to reach the chloroplast of a cell as this is where starch is produced.

METHOD:

We transformed our binary vector constructs (see results “Making constructs to express putative acyl-transferases in plant chloroplasts” for the details of these constructs) containing the acyltransfeases with an N-terminal transit peptides and C-terminal fluorescent reporters into Agrobacterium tumefaciens by electroporation (see Protocols - here). Colonies were checked by PCR before liquid cultures were grown from individual colonies for infiltration of plant leaves (see Protocols - here). Finally, infiltrated leaves were examined by confocal microscopy to determine the subcellular localisation of the recombinant protein.

Determining if the expression of acyl-transferases interferes with starch accumulation in plants

AIM:

To compare the starch content of leaves infiltrated with A.tumefaciens strains carrying constructs expressing our putative acyltransferases. A potential problem of using putative acyl transferases is that they will use the Carbon 4 position that the starch synthases usually extend starch molecules from causing a reduction in the starch content. A simple test was to confirm that normal quantities of starch were being produced.

METHOD:

We transformed our binary vector constructs (see results “Making constructs to express putative acyl-transferases in plant chloroplasts” for the details of these constructs) containing the acyltransfeases with an N-terminal transit peptides into Agrobacterium tumefaciens by electroporation (see Protocols - here). Colonies were checked by PCR before liquid cultures were grown from individual colonies for infiltration of plant leaves (see Protocols - here) (Figure 1).

The infiltrations were done on three plants. We infiltrated: A. tumefaciens with no construct (control), non-infiltrated leaves (control) along with strains of A. tumefaciens that contained the binary vectors. The plants were of the same age, same species, and were grown in the same conditions.

After infiltration, the plants were left in a normal light/dark cycle at room temperature for 24 hours before being put in the dark for 24 hours. In the dark the plants would break down their already the stores of starch in their leaves. After the dark treatment, two controls, two empty leaves, and the first set of acylation enzyme infiltrated leaves were decolorized to remove the green chlorophyll and stained with iodine, which stains starch granules. A duplicate set of experiments was allowed to grow in the light for the rest of the day to build up new stores of starch. These were then assayed in the same manner to determine if expression of the putative acyltransferases interfered with starch accumulation.

RESULTS:

Colonies of A. tumefaciens were screened by PCR to confirm the presence of the binary construct (Figure 1).

Figure 1: The gel lanes are as follows – 1 = Ladder, 2 = BBa_K1618033 control, 3&4 = BBa_K1618033 Agrobacterium, 5 = BBa_K1618035 control, 6&7 = BBa_K1618035 Agrobacterium, 8 = Ladder, 9 = BBa_K1618036 control, 10&11 = BBa_K1618036 Agrobacterium, 12 = BBa_K1618034 control, 13&14 = BBa_K1618034 Agrobacterium. The comparison between the agro-transformations and the sequence confirmed control suggests that the cloning was successful.

After 24 hours in the dark the leaves from all samples were free of starch (Figure 2a). This allowed us to determine the amount of starch accumulated in the following 24 hours. Leaves sampled after 24 hours in the light all stained dark with iodine. There is no difference in the amount of staining observed in samples from leaves infiltrated with the A. tumefaciens strains that contained the binary vectors expressing putative acyl transferases as compared to the control samples (Figure 2b).

Figure 2: Iodine staining of leaves after (a) 24 hours dark treatment, and (b) dark treatment and 8 hours light treatment. Staining from dark treatment (a) are yellow, indicating no starch is present. While there are black spots observed, it was found that these are due to damage from infiltration rather than starch. Iodine staining from dark and light treatment (b) stained black, indicating presence of starch. The black staining doesn’t appear to differ between the different leaves and their conditions, suggesting that the infiltrations and the enzymes do not affect starch production in leaves.

These results indicate that neither the infiltration process or the expression of putative acyl-transferases has an impact on the production of starch.

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