Our Action Plan

In our project this year, we aim to incorporate the carotenoid biosynthesis pathway in E. coli and produce different coloured pigments. Potential candidates are lycopene (red), beta-carotene (orange), and zeaxanthin (yellow). Since these three compounds are sequential in the biosynthetic pathway, we hope to demonstrate colour changes from red to orange to yellow, by controlling expression of the enzymes involved in conversion.

Our action plan is as follows:

1. Test the activity of K152005 (CrtEBIY).
2. Test pigment production in E. coli strain MG1655 and its derivative (up-regulation in precursors).
3. Construct composite biobrick containing CrtE, CrtB and CrtI (i.e. CrtEBI) and test its activity.
4. Transform E. coli with CrtEBI and CrtY. Test if activation of CrtY can cause colour changes in vivo.
5. Characterise our composite biobricks and the colour output.
6. Test compatibility with biobricks from previous iGEM teams and see if we can convert their outputs into pigment production.
Our Action Plan (Updated regularly). Green: completed. Yellow: Work in progress. Blue: Partially completed. Red: Planning.

Test activity of K152005

Close-up of K152005 in TOP10

We wish to test the feasibility of incorporating the carotenoid biosynthetic pathway into E. coli, which does not produce carotenoids naturally. K152005 contains the enzymes for beta-carotene formation, and is a quick way to verify our concept.

We transformed E. coli (TOP10) using K152005 from the 2009 Distribution Plate (Week 2). After incubation for about 3 days, some light yellowish pigmention was visible. The long incubation time may be due to the fact that K152005 does not contain promoter. Although only small amount of yellow pigment was observed*** (instead of the expected orange colour), this gave us confidence that the carotenoid biosynthetic pathway was working in E. coli. ***Later experiments showed that the yellow pigment was probably not beta-carotene. Please refer to "Characterisation of Biobricks and colour output" for more details.

Mapping of Biobrick K152005 (update):

Bildschirmfoto-K152005(CrtEBIY) updated.ape Map.png

We will then put K152005 under a strong promoter (R0011, constitutive) to see if stronger colour production can be achieved.

Test pigment production in E. coli MG1655 and its derivative

Pathway from pyruvate and G3P to FPP. Figure from Yuan et al. 2006.

The first compound in the carotenoid biosynthetic pathway, farnesyl pyrophosphate (FPP), derives ultimately from pyruvate and glyceraldehyde 3-phosphate (see figure on the right). The rate of carotenoid synthesis is limited by the rate of supply of FPP.

Certain strains of E. coli, e.g. MG1655, have been genetically engineered to up-regulate the enzymes involved in the pathway leading to FPP, thus increasing FPP supply and enhancing carotenoid synthesis. In Yuan et al., plasmid pPCB15 was used as reporter to assess the effect of such up-regulation. pPCB15 contains genes CrtEXYIB derived from Pantoea stewartii (homologous to the Biobricks available, which are derived from Pantoea ananatis) and produces orange beta-carotene as the main coloured product. The results showed that upregulation of enzymes upstream of FPP led more beta-carotene production.

Map of plasmid pPCB15.

In our project, we wish to compare carotenoid pigment production in E. coli strains TOP10, MG1655 and its derivative. Due to greater supply of FPP, we will expect higher level of carotenoid production in MG1655 and its derivative than in TOP10. Working on MG1655 and its derivative makes it easier to assess the effects of Biobricks on carotenoid synthesis, and allows us to develop and refine methods for pigment measurement and characterisation, which can then be applied on TOP10.

We have access to the original MG1655 ("Parent") and its derived strain, MG1655 PT5-dxs PT5-idi PT5-ispB PTe-ispDF ("Roche"). We first transformed these two strains with pPCB15 for orange beta-carotene production (Week 3). pPCB15 was also transformed into TOP10 to see if it would work in a strain with normal FPP flux. pPCB15 has chloramphenicol-resistance selection marker so the transformed bacteria were grow on media with chloramphenicol.

On agar plates, pPCB15 produced yellow pigments in all three strains, with increasing intensity in the order: TOP10 < original MG1655 < MG1655 PT5-dxs PT5-idi PT5-ispB PTe-ispDF (photo below). The results indicated that it is feasible to introduce carotene synthesising pathway in E.coli, and that even in strains with normal FPP flux (i.e. TOP10), the yellow pigment production is still relatively fast (overnight incubation) and easily visualised. However, as mentioned above, since the effects in MG1655 and its derivative are much stronger and easier to assess, we used them as a tool to prove our concepts and test our Biobricks.

Top row from the left: pPCB15 in MG1655-PT5 ("Roche"), pPCB15 in TOP 10, pPCB15 in MG1655 ("Parent"). Bottom row from the left: K152005 in TOP10, R0011 in TOP10 (negative control).

Construct composite Biobrick CrtEBI

We wish to produce the first coloured compound in the pathway, lycopene (red), which can then be converted into other colours by subsequent enzymatic reaction.

In order to “pause” at lycopene, we need enzymes CrtE, CrtB and CrtI only. Currently in the registry there is no available Biobrick containing CrtEBI, so we decide to construct our own.

After transforming E. coli with K118014 (rbs+CrtE), K118006 (rbs+CrtB) and K118005 (rbs+CrtI), we construct composite biobrick by ligating the three genes together.

Plan mapping of CrtEBI:

Bildschirmfoto-CrtEBI.ape Map.png

Test colour changes by CrtY

Characterisation of Biobricks and colour output

Carotene: We wish to not only visually identify yellowish/orange pigment formation, but also quantify the amount of beta-carotene produced. To this end, we performed carotene extraction with acetone using protocol described in literature and took absorbance measurement using Omega Microplate Readers (BMG-Labtech).

As a pilot project to test out the carotene measurement protocol, we prepared overnight cultures for pPCB15 in original MG1655, pPCB15 in MG1655 PT5-dxs,idi,ispBDF, pPCB15 in TOP10, K152005 in TOP10 and R0011 in TOP10 (as negative control). Absorbance spectrum measurements were taken, first on intact cells, and then on carotene extract in acetone. The results indicated that absorbance measurements on intact cells were unable to demonstrate the characteristic absorbance peak of beta-carotene at 450 nm. In contrast, absorbance measurement on carotene extract in acetone showed clear peak at 450nm, which was a much better indicator of beta-carotene production. Furthermore, the visually identified "yellow pigment" in K152005 was probably not beta-carotene. This could not have been distinguished using visual inspection along. Given these results, we decided to use carotene extraction in acetone and absorbance at 450 nm for future quantification of amount of carotene.

The colours of the acetone extracts were shown below:

Cambridge simingpic.jpg

Results of the Microplate Readers (left: absorbance spectrum of intact cells; right: absorbance spectrum of acetone extracts).See Week 3 Notebook for detailed analysis of the results.

Cambridge Intact cell.png Cambridge Carotene extraction.png

Test compatibility with other biobricks

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