Difference between revisions of "Team:NRP-UEA-Norwich/Results/Prebiotic"
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| − | <p class="space10">We used Golden Gate Cloning and the Plant Standard Syntax<sub><a data-id="ref" class="scroll-link" style = "color: #002bb8;">1</a></sub> to make our constructs. We used 35s promoter from Cauliflower Mosiac Virus (<a href=http://parts.igem.org/Part:BBa_K1467101"style="color:#002bb8;">BBa_K1467101</a>), to drive constitutive expression. We made a chloroplast transit peptide (New part - <a href=http://parts.igem.org/Part:BBa_K1618028"style="color:#002bb8;">BBa_K1618028</a>) in an N-terminal translation fusion with the acyl transferases for the protein to reach the chloroplast, where starch is produced. | + | <p class="space10">We used Golden Gate Cloning and the Plant Standard Syntax<sub><a data-id="ref" class="scroll-link" style = "color: #002bb8;">1</a></sub> to make our constructs. We used 35s promoter from Cauliflower Mosiac Virus (<a href="http://parts.igem.org/Part:BBa_K1467101"style="color:#002bb8;">BBa_K1467101</a>), to drive constitutive expression. We made a chloroplast transit peptide (New part - <a href="http://parts.igem.org/Part:BBa_K1618028"style="color:#002bb8;">BBa_K1618028</a>) in an N-terminal translation fusion with the acyl transferases for the protein to reach the chloroplast, where starch is produced. |
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Revision as of 20:09, 17 September 2015
RESULTS - PREBIOTIC
Making constructs to express inducible branching and debranching enzymes in E. coli
AIM:
We aimed to ligate the E. coli glycogen metabolic genes GlgX and GlgB into the standard pSB1C3 plasmid vector with and without an IPTG-inducible promoter. GlgX is a glycogen debranching enzyme which cleaves α-1,6 glycosidic linkages in glycogen to remove branches. GlgB is a glycogen branching enzyme which cleaves α-1,4 glycosidic linkages and re-anneals the cleaved linkage back onto the main chain through an α-1,6 linkage, therefore creating a branch. Creating parts with the promoter allows us to investigate the effect of the expression of these enzymes on E. coli glycogen in an inducible system. Basic parts with just the enzyme coding sequences were also generated so that they can be used to build other composite parts in the future.
METHOD:
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:
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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.
METHOD:
We used Golden Gate Cloning and the Plant Standard Syntax1 to make our constructs. We used 35s promoter from Cauliflower Mosiac Virus (BBa_K1467101), to drive constitutive expression. We made a chloroplast transit peptide (New part - BBa_K1618028) in an N-terminal translation fusion with the acyl transferases for the protein to reach the chloroplast, where starch is produced.
In order to confirm that the transit peptide was functional we made a second set of constructs that had a fluorescent reporter in a C-terminal translational fusion.
To deliver the constructs to plants we needed to use Agrobacterium tumefaciens as a shuttle chassis. This meant parts needed to be assembled into a binary vector with origins of replication for both E.coli and A. tumefaciens. In order to submit the parts to the registry we also assembled parts into the MoCloFlipper pSB1C3 that accepts Golden Gate Parts into the pSB1C3 backbone.
All cloning was done according to the Golden Gate one-step Digestion-Ligation Protocol provided on our protocols page here.
Results:
Successful assembly into the binary vector.
Putative clones were screened by colony PCR (see Protocols for method - here) using primers that flank the insertion sites (Figure 1).
Figure 1: The gel lanes are as follows – Ladder, 1&2 = MAO, 3&4 = MAO + YFP 5&6 = RV3034c, 7&8 = RV3034c + YFP, 9&10 = RV3037c, 11&12 = RV3037c + YFP, 13&14 = RV3030, 15&16 = RV3030 + YFP, ladder. The gel image above indicated that the complete transcriptional units were successfully cloned.
A single colony for each construct was mini-prepped and sequenced before progression to transformation into A. tumefaciens.
Successful assembly into pSB1C3.
Putative clones were miniprepped (see Protocols for method - here) and screened by digestion with NotI (Figure 2) and also with EcoRI/PstI (Figure 3) to confirm that cloning was successful and that our constructs had no internal BioBrick restriction sites.
Figure 2: Gel lanes are as follows - Ladder, 1&2 = MAO, 3&4 = MAO + YFP, 5&6 = RV3034c, 7&8 = RV3034c + YFP, 9&10 = RV3037c, 11&12 = RV3037c + YFP, 13&14 = RV3030, 15&16 =RV3030 + YFP, Ladder. These indicate that there are no unwanted internal Not1 restriction sites within our constructs.
Figure 3: Gel lanes are as follows - Ladder, 1&2 MAO + GFP, 3&4 = RV3034c + GFP, 5&6 = RV3037c + GFP, 7&8 = RV3030 + GFP, 9&10 = MAO + GFP, 11&12 = RV3034c + GFP, 13&14 = RV3037c + GFP, 15&16 = RV3030 + GFP. Lanes 1-8 were digested with Not1 while lanes 9-16 were digested with EcoR1/Pst1. The results indicate that the new tags allow our constructs to be BioBrick compatible.
A single colony for each construct was mini-prepped and sequenced before shipping to the registry.
References
1. Patron et al. (2015) Standards for Plant Synthetic Biology: A Common Syntax for Exchange of DNA Parts doi: 10.1111/nph.13532/full
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.
RESULTS:
Colonies of A. tumefaciens were screened by PCR to confirm the presence of the binary construct (Figure 1).
Figure 1: Gel lanes are as follows – (Top row) Ladder, 1-3 = BBa_K1618029 (MAO enzyme), 4-6 = BBa_K1618031 (RV3034c enzyme), 7-9 = BBa_K1618032 (RV3037c enzyme), 10-12 = BBa_K1618030 (RV3030 enzyme), Ladder. The comparison between the agro-transformations and the sequence confirmed control suggests that the cloning was successful.
Leaves that had been infiltrated with A. tumefaciens strains containing our assembled binary constructs were images using an SP5 Leica confocal microscope (Figure 2). We used two channels: the first excites chlorophyll, which is shown in red and the second excites the fluorescent fusion protein (shown in yellow).
Figure 2: Constructs BBa_K1618029-032 contain a yellow fluorescent protein, as well as a chloroplast transit peptide. These are confocal microscopy images of the constructs infiltrated into Nicotiana benthamiana, in which the red structures are the chlorophyll within the chloroplast, and the yellow is the fluorescent fusion protein expressed from constructs (a) BBa_K1618029 (MAO enzyme), (b) BBa_K1618031 (RV3034c enzyme), (c) BBa_K1618032 (RV3037c enzyme), and (d) BBa_K1618030 (RV3030 enzyme).
Our results confirm that the putative acyltransferases are successfully localised to the chloroplasts of the plants.
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: Gel lanes are as follows – Ladder, 1-3 = BBa_K1618033, 4-6 = K1618035, Ladder, 7-9 = K1618036, 10-12 = K1618034. 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.
