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From 2011.igem.org

Results for Ranaspumin

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We were successful in cloning our Ranaspumin BioBrick into the pET23 vector, yet our attempts to ligate it into the submission vector yielded negative results. Submitting it as a theoretical BioBrick became a top priority.

Results for Latherin

E. coli expressing latherin under the control of a pBAD promoter. The presence of a significant level of foam compared to the negative controls clearly demonstrates its surfactant properties.

We aimed to build a series of constructs of OmpC, OmpF and Bluf promoters, connected to eYFP (BBa_I13001) and a constructed RFP reporter, to determine whether the light responsive promoters work at all, and eventually characterise them.

This work would serve as a proof of concept that expression of our novel biobricks via light is feasible.

Materials and Methods:

List of biobricks used:

- B0030, Strong RBS, Ap+

- B1006, Strong double terminator, Ap+/Kn+

- E1010, RFP, Kn+

- I13002, Weak RBS – eYFP – double terminator (sYFP), Ap+

- I13001, Strong RBS – eYFP – double terminator (wYFP), Ap+

- K238013, Bluf promoter, Ap+

- R0084, OmpF promoter, Ap+

- R0082, OmpC promoter, Ap+

- K225000, GlrN (green light receptor), Cm+

Ap+ refers to Ampicillin resistance

Kn+ refers to Kanamycin resistance

Cm+ refers to Chloramphenicol resistance

Cells

- Top10 chemically competent cells from Invitrogen

- Δ EnvZ CP919 E.Coli cells (BBa_V1012)

Top10 cells were initially transformed with each of the biobricks, overnighted and miniprepped.

CP919 cells were made competent and stored until ready to be transformed with the completed constructs.

RFP assembly method:

The RFP biobrick was digested with EcoRI-SpeI and ligated to the strong double terminator, previously cut with EcoRI-XbaI. The obtained ligation was then inserted into Top10 cells and selected on agar/kanamycin plates.

At the same time a library of Promoters/RBS was made.

(check here our toolbox of promoter-RBS!)

The RFP-Terminator construct was then digested with XbaI-PstI and ligated to each of the available Promoter/RBS, after previous digestion with SpeI-PstI.

The obtained Promoter-RBS-RFP-Terminator constructs were then digested with EcoRI-PstI and ligated into the submission vector.

Edinburgh biobricks:

We received the following parts from the Edinburgh team:

- EDIN 1, lac promoter with phycocyanobilin biosynthesis pathway - based on BBa_K322122

- EDIN 2, corrected cph8 + RBS without promoter - based on BBa_K322124

- EDIN 3, OMPC promoter with YFP

- EDIN 4, cph8 + pOmpC-eYFP - based on BBa_K322126

- EDIN 5, Trp Promoter with mCherry (Lov-Tap reporter)

- EDIN 6, LOV-Tap plus Ptrp mCherry

- EDIN 7, LOV-Tap

- EDIN 8, cph8 plus P-ompc lac Z

These parts were submitted onto the registry but they have failed to yield any results. Edinburgh found that there were errors in the DNA sequences in these parts and attempted to correct them. They gave the parts to us in the pSB1C3 and we have pursued their characterisation and included the promoters in our library of promoters and ribosome binding sites. We did not find any of these parts to work. We have sequenced the parts given to us by Edinburgh as our contribution towards improving them. The cph8+ pOmpC promoter in pSB1C3, LOV-Tap+ trp promoter-mCherry in pSB1C3, corrected light sensor cph8 with RBS but no promoter, and cph8 plus pOmpC-lacZ in pSB1C3 were the ones sequenced by our team to contribute to the Edinburgh 2010 registry pages.

Results:

In the first place we wanted to test the reliability of our constructs and we wanted to see with our own eyes to what extent is gene expression affected by a weak or strong ribosome binding site.

We then grew the cell cultures containing all the component to detect light, in dark and light and compared the fluorescence to determine the functionality of the light responsive system.

Figure 2 above shows how the strength of the RBS influences the levels of YFP expression in Top10 E.coli cells. These cells are lacking the machinery required to detect red light (phycocyanobilin path. + cph8) therefore the expression at the OmpF promoter is constitutive. The intensity of the YFP is reflected by the strength of the RBS. This shows how our constructs can be used to optimise expression levels in multi-step pathways to maximise carbon-flux and avoid unnecessary build-up.

Figure 3: EcoRI-PstI digest of OmpF-sYFP and OmpF-wYFP(uncut sample followed by double digest). 1 Kb ladder is on the right hand side.

The gel electrophoresis of the two version of the OmpF-YFP construct show that the cells we used for microscopy contained the correctly assembled construct.

Figure 4

This set of pictures on Figure 4 show incomplete light responsive constructs.

Unfortunately we realised too late that the EDIN1+2 ligation was in fact either just an EDIN1 which reannealed on itself or a dimerised plasmid backbone, byproduct of the ligation reaction.

The controls show a constitutively expressed RFP under control of an OmpF promoter lacking the light sensing machinery.

The negative control shows the green haze at the air-culture border under the coverslip. We suggested that this type of green fluorescence is due to the LB media in which the cells were cultured.

The series of images shown here provide unconsistent results: - any RFP under control of OmpC is supposed to be transcriptionally silent or very weakly expressed. The RFP fluorescence could be due to an RFP on the backbone of the GlrN vector, but we’re unable to check that on the registry. - the OmpF-RFP containing culture contained also a wrong EDIN1+2 ligation thus the red fluorescence was expressed in a constitutive manner. We are anyway unable to show that the fluorescence was due to a correctly assembled construct.

Positive results for Bluf-sYFP

Figure 5

Figure 5: Digest of Bluf-sYFP with EcoRI-PstI(uncut sample followed by double digest). 1Kb ladder on the right handside.

The Bluf-sYFP grown in light (5th picture from the top in Table 3) shows higher YFP expression levels than the same culture grown in darkness (6th from the top). The gel electrophoriesis on the left handside shows the right band pattern for Bluf-sYFP after digestion with EcoRI-PstI, proving that this behaviour was due to the presence of a correct construct. Unlike Bluf-sYFP the results for OmpF-sYFP grown in light and dark are inverted, and both samples contain a non-functional red light receptor. The increase in fluorescence can be simply due to an higher density of cells in the sample grown in dark or by an advanced stage of growth (the culture wasn't on a shaker for several hours and lumps of cells are visible).

The OmpC samples glow of a faint red regardless of the absence on any RFP in the construct. again, we can speculate about the presence of such an RFP on the backbone of the GlrN vector.

Discussion

The experiment worked as expected only for the Bluf-sYFP construct, and unfortunately we didn’t manage to produce a Bluf-RFP construct to confirm our results.

The OmpF and OmpC promoters didn’t behave as expected for a number of reasons:

- the cells didn’t contain all the machinery required to detect light. GlrN also required the presence of the phycocyanobilin chromophore to work so we’re not surprised that OmpC wasn’t responsive to light.

- the phycocyanobilin pathway present in cells containing OmpF-reporter construct lacked the cph8 receptor.

- All the ligations of OmpC /reporter construct didn’t produce a positive result. The reason why we failed to ligate the two was probably due to the large amounts of DNA that went lost when extracting the digested insert from its agarose gel, and our failure to balance the amounts of insert and backbone to be used in the ligation reaction.