Part:BBa_K302012:Experience

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Applications of BBa_K302012

Characterisation by Team Newcastle 2010

We integrated our part into the Bacillus subtilis 168 chromosome at amyE (using the integration vector pGFP-rrnB) and selected for integration by testing for the ability to hydrolyse starch. Homologous recombination at amyE destroys endogenous expression of amylase. Colonies that are not able to break down starch on agar plate do not have a white halo when exposed to iodine.

The part was co-transcribed with gfp fluorescent marker by transcriptional fusion after the yneA coding sequence.

We characterised the part first without, and then with, LacI repression (using the integration vector pMutin4 to integrate lacI into the Bacillus subtilis 168 chromosome). When testing the part under LacI repression cells were induced with IPTG for two hours.

Table1:

Stats:	168	yneA	pMutin4 0 μM IPTG	pMutin4 1 μM IPTG
Average:	1.34 μm	3.53 μm	1.74 μm	3.19 μm
Max:	2.30 μm	6.00 μm	3.62 μm	9.77 μm
Min:	0.55 μm	1.31 μm	0.88 μm	1.14 μm
Median:	1.33 μm	3.27 μm	1.62 μm	2.66 μm
Standard Deviation:	0.32 μm	1.01 μm	0.80 μm	1.56 μm

Figure 1:

Distribution of cell lengths is not normal, so the mean is misleading; we are reporting the median instead.

Figure 1: shows statistics for populations of cells overexpression of the yneA construct (ΔamyE:pSpac(hy)-oid::yneA(cells with YneA construct but no inhibitory regulation) ) leads to a longer cell length compared with our control Bacillus subtilis 168. pMT4_0.0: YneA construct in pMutin4 vector with inhibition and no IPTG (ΔamyE:Pspac(hy)-oid::yneA::pMutin4) pMT4_1.0: YneA construct in pMutin4 vector with inhibition and 1.0 μM IPTG (ΔamyE:Pspac(hy)-oid::yneA::pMutin4)

with inhibition cell lengths are comparable to Bacillus subtilis 168 at 0μM IPTG and longer with IPTG induction.

Figure 2:

Figure 2: Bacillus subtilis 168 cells (left),Bacillus subtilis expressing yneA(centre) and Bacillus subtilis overexpressing yneA(right)

The images we have taken this data from had very different numbers of cells, so the cells counts are misleading therefore we are reporting the proportions of cells at a given length.

Figure 3:

Figure 3 shows the percentage of cells at different lengths (μm) uninduced

Figure 4:

Figure 4:Bacillus subtilis 168 cells (left) and non-induced cells (right)

Figure 5:

Figure 5: shows the percentage of cells at different lengths(μm)induced at 0.2mM IPTG

Figure 6:

Figure 6: Bacillus subtilis 168 cells (left) and cells induced at 0.2mM IPTG (right)

Figure 7:

Figure 7: shows the percentage of cells at different lengths (μm) induced at 1mM IPTG

Figure 8:

Figure 8: Bacillus subtilis 168 cells (left) and cells induced at 1mM IPTG(right)

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