Team:Manchester/Description/mechanism2

Manchester iGEM 2016

Mechanism 2

Inducible Gene Switch


Mechanism 2 overview diagram




How it works?


Mechanism part 1 Figure 1
Figure 1: Schematic representation of the plasmid constructs used in our project.

alcR

alcR is a positive regulatory gene in the ethanol regulon of filamentous fungus Aspergillus nidulans (A.nidulans). It encodes a protein that acts as a transcription factor which would bind to its target promoters alcA and aldA. The expression of the downstream gene of alcA promoter is strongly induced through the positive transcriptional regulator AlcR protein by various substrates such as ethanol and threonine. For our project, we were interested in the ability of the AlcR protein, under the influence of ethanol, to initiate transcription of chromoproteins by binding to specific sites on the alcA promoter [1]. The chromoproteins used were from previously characterised BioBricks by 2013 iGEM Uppsala-Sweden: amilCP with RBS (BBa_K1033930) and spisPink with RBS (BBa_K1033925).


AlcR is also known as a zinc binuclear cluster activator as it contains a DNA-binding domain belonging to the C6 zinc binuclear cluster family. AlcR is unique in that it can bind to symmetric and asymmetric DNA sites with the same apparent affinity and also bind to single site with high affinity [2]. Our project focuses on the ability of AlcR to bind to its binding sites on the alcA promoter. alcR is a new BioBrick we have characterised this year (BBa_K2092004).




alcA promoter (PalcA)

PalcA is one of the strongest inducible promoters in A.nidulans and is widely used to overexpress proteins [2]. Based on our references, we found that the number and the position of the AlcR binding sites on PalcA are crucial for the strength of transcriptional activation of PalcA. Hence, we took the factors into account when deciding which PalcA variants to use for our system. There are evidences that show two sites, either in direct or inverted orientation, are necessary for full transcriptional activation of PalcA [3].


Our PalcA variant ((PalcA(var)) was inspired by a research done in creating a functional, chemically inducible gene switch for monocotyledonous plant sugar cane [3] and Escherichia coli [4]. PalcA is the native variant which is a previously characterised BioBrick by 2011 iGEM DTU-Denmark (BBa_K678001). We have improved this BioBrick by adding several missing restriction sites on the Prefix and Suffix (BBa_K2092002). PalcA(var) is a new BioBrick we have characterised this year (BBa_K2092003).

Mechanism 2 Part 1 Figure 2
Figure 2: Schematic representations of PalcA variant constructs adapted from [1]. Binding site “a” consists of two direct tandem repeats, “b” consists of two inverted tandem repeats and “c” consists of three half sites with both direct and inverted tandem repeats.

PalcA and PalcA(var) differ in the binding sites for AlcR. PalcA has binding sites abc while PalcA(var) contains only binding sites bc. Binding site a contains two direct tandem repeats; binding site b, a palindromic target, contains two inverted tandem repeats while binding site c consists of three half sites with both direct and inverted tandem repeats. The binding sites abc have been previously localized in the PalcA by footprinting experiments and it has also been shown that each AlcR target in the PalcA contributes differently to the activation of the downstream protein expression [3].




What have we achieved over the summer?


Mechanism 2 Part 2 list

Plasmid verification

All parts obtained from previous iGEM teams were first transformed. The samples were then verified through restriction enzyme digest using NEB enzymes before being used to assemble our desired plasmids for the project.

Figure 1 Figure 3: 1% Agarose gel showing restriction enzyme digest results. Band sizes expected can be seen in Table 1.
table 1 Table 1: Table outlining the details of Figure 3.The restriction enzymes used and band sizes expected are shown.



Based on the digest, it was concluded that all samples except CP2 and PalcA gave us a positive conformation of the plasmid. We repeated the digest for CP2 and PalcA with several other enzymes and managed to get a positive verification of CP2 but not PalcA.




Figure 2
Figure 4: 1% Agarose gel showing restriction enzyme digest results. Band sizes expected can be seen in Table 2.
Table 2 Table 2: Table outlining the details of Figure 4. The restriction enzymes used and band sizes expected are shown.



We then proceeded to send two samples of PalcA for sequencing to confirm that the part obtained is correct. The sequencing results confirmed that two restriction sites – XbaI and SpeI are absent from the BioBrick Prefix and Suffix.




Table 3
Table 3: Table outlining the sequencing results of PalcA. It can be concluded that restriction sites XbaI and SpeI are absent.



Hence, we decided to add another element to our project – an improved BioBrick by adding the two missing restriction sites on the Prefix and Suffix.




Constitutive Promoter (CP) Characterization


Our choice of constitutive promoters come from the constitutive promoter family created by 2006 iGEM Berkeley. We tested two of the variants, CP1 (BBa_J23100) and CP3 (BBa_J23110 ) that are present in the BBa_J61002 backbone that contains red fluorescent protein (rfp) downstream of the promoter. We could not test CP2 (BBa_J23119) as it came in a psB1A2 backbone that does not contain rfp.


Figure 1 Figure 5: Schematic representation of CP1 plasmid (BBa_J23100) adapted from Snapgene.
Figure 2 Figure 6: Schematic representation of CP3 plasmid (BBa_J23110 ) adapted from Snapgene.

We performed an rfp quantification by comparing three technical and two biological replicates of CP1 and CP3. Cultures were first normalised to an appropriate OD and then 200µl of the culture was added to each well. This is to ensure that the initial cell density is uniform for all samples.

An aTC inducible Kiesling vector, pBb2K, was used as a positive control. The vector was induced at mid-log phase when OD 600 = 0.4 by adding 1µl of 1mM aTC.


Figure 7: Image of the 96-well plate used for the rfp quantification. Quantification was done using a FLUOstar Omega (BMG Labtech) plate reader. OD600 and rfp (λ= 544nm) was measured every 30 minutes over a period of 24 hours. During idle time, the plate was shaken at 500rpm. The plate reader maintained a constant temperature of 37oC throughout the duration of quantification.



Chromoprotein Characterization


The aim of this chromoprotein characterization is to show that under the control of the same promoter, both of our chosen chromoproteins: amilCP with RBS (BBa_K1033930) and spisPink with RBS (BBa_K1033925), are clearly visible at the same time point. To show this, we first inserted the chromoproteins downstream of our choice of constitutive promoter (BBa_J23110). We prepared overnight cultures from a single colony and a dilution was made the next morning to normalize it to an OD600 of 0.65. This is to ensure the samples have the same initial cell density. 20µl of each sample was then plated onto a chloramphenicol plate. Pictures were taken at various time points to monitor colour visibility.




It can be concluded that both chromoproteins produce visible colours at the same time point and hence is suitable to use for our project.




Improved PalcA BioBrick


part 6 figure 1
Figure 8: Schematic representation of the PCR method used to add the missing restriction sites on the BioBrick Prefix and Suffix.

PalcA (BBa_K678001) is a BioBrick requested from the iGEM HQ as it was unavailable in the 2016 kit. Sequencing results confirmed the absence of two restriction sites – XbaI and SpeI on the BioBrick Prefix and Suffix. As these two sites are crucial in the assembly of our composite parts, we designed primers to add the two missing restriction sites.


part 6 table 1
Table 4: Table showing the primer sequence used to add the missing restrictions sites on the BioBrick Prefix and Suffix.




References

  • Panozzo, C., Capuano, V., Fillinger, S. and Felenbok, B. (1997). The zinc binuclear cluster Activator AlcR is able to bind to single sites but requires multiple repeated sites for synergistic activation of the alcA gene in Aspergillus nidulans. Journal of Biological Chemistry, 272(36), 22859–22865.
  • Felenbok, B., Sequeval, D., Mathieu, M., Sibley, S., Gwynne, D.I. and Davies, R.W. (1988). The ethanol regulon in Aspergillus nidulans: Characterization and sequence of the positive regulatory gene alcR/i>. Gene, 73(2), 385–396.
  • Kinkema, M., Geijskes, R.J., Shand, K., Coleman, H.D., De Lucca, P.C., Palupe, A., Harrison, M.D., Jepson, I., Dale, J.L. and Sainz, M.B. (2013). An improved chemically inducible gene switch that functions in the monocotyledonous plant sugar cane. Plant Molecular Biology, 84(4-5), 443–454.
  • Hemmati, H. and Basu, C. (2015). Transcriptional analyses of an ethanol inducible promoter in Escherichia coli and tobacco for production of cellulase and green fluorescent protein. Biotechnology & Biotechnological Equipment, 29(6), 1043–1052.

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