Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing. The culture was plated from the agar stab and also a liquid culture set up.

Protocols Used: Plating, Overnight cultures

Results: N/A

Next Steps: Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.

Aim of experiment: To purify BBa_K1058008 for sequence confirmation.

Protocols Used: Miniprep plasmid purification, DNA sequencing

Results Sequencing: Figure 1

Next Steps: PCR of promoter region (pChr) and repressor region (ChrB) of BBa_K1058008 for storage in individual biobricks.

Aim of experiment: To break down BBa_K1058008 into the promoter region pChr, and the repressor gene, ChrB, while adding standard prefix and suffix to each. Furthermore optimisation of first 15 codons of ChrB in order to increase the overall rate of transcription. Restriction digest of the respective ends and subsequent ligation of pChr and ChrB into pSB1C3 for submission to registry.

Protocols Used: PCR, Gel extraction, Restriction Digest

Results: Figure 2

Next Steps: Repeat of PCR of ChrB. Purification of the Plasmid, containing BBa_K1058008 and confirmation of size and sequence.

Aim of experiment: To repeat PCR of ChrB for subsequent ligation into pSB1C3. Ligation of pChr and ChrB into pSB1C3. Transformation of each into a non-pathogenic lab strain of Escherichia coli. Overnight culture of E. coli DH5-alpha, containing GFPmut2, as a fluorescent reporter for our chromate sensing system

Protocols Used: PCR-amplification, Gel extraction, Restriction digest, Ligation, Overnight cultures

Results: Figure 3

2 bands were extracted from the gel for further characterisation, the strongest band, hence called ChrBs, and the band directly above, hence called ChrBw. pChr, ChrBs, and ChrBw were ligated into pSB1C3. pSB1C3-pChr, pSB1C3-ChrBs, and pSB1C3-ChrBw were transformed into E. coli MC1061.

Next Steps: Sequence and size confirmation of pChr-pSB1C3, ChrBs-pSB1C3, and ChrBw-pSB1C3. Ligation of pChr to GFP coding sequence for construction of a reporter plasmid.

Aim of experiment: Purification of GFPmut2 from overnight cultures. Overnight cultures of recombinant E. coli MC1061, containing pSB1C3-pChr, pSB1C3-ChrBs, and pSB1C3-ChrBw respectively for further size and sequence confirmation. Overnight culture of pUniprom for subsequent ligation of ChrB

Protocols Used: Miniprep, PCR-amplification, Gel extraction, Overnight cultures

Results: Figure 5

Next Steps: Repeat of amplification of ChrB. Sequence and size confirmation of pSB1C3-pChr, pSB1C3-ChrBs, and pSB1C3-ChrBw.

Aim of experiment: To purify overnight cultures for size and sequence confirmation. Repeat of PCR amplification of pChr and ChrB. Restriction digestes of all parts in order to produce compatible sticky ends.

Protocols Used: Miniprep, PCR amplification, Restriction digest, Sequencing

Restriction digest of gel purified PCR-products of pChr, ChrB, and GFPmut2, using EcoRI/SpeI, BamHI/HindIII, and XbaI/PstI respecitvely.

Results:

Figure 6 - Size confirmation of pChr and ChrB.

Figure 7 - Sequence confirmation of pChr

Figure 8 - Sequence confirmation of ChrB.

Next Steps: Purification and digest of pUniprom for insertion of ChrB.

Aim of experiment: To prepare pUniprom for insertion of ChrB by digesting it with BamHI and HindIII. To amplify ChrB version with first 15 codons optimised for insertion into pUniprom.

Protocols Used:

Restriction digest and Gel extraction of pUniprom

PCR and Gel extraction of optimised ChrB

Results:

Figure 9 Gel with pUniprom

Figure 10 Gel with ChrB

Next Steps: Ligation of ChrB and codon optimised ChrB into pUniprom.

Aim of experiment: Ligation of ChrB and optimised ChrB into pUniprom via BamHI and HindIII restriction sites. Ligation of pChr and GFPmut2 to each other via SpeI/XbaI-link and into pSB1C3 via EcoRI and PstI restriction sites.

Protocols Used: Ligations

Results: N/A

Next Steps: Transformation of abovementioned ligations.

Aim of experiment: Transformation of pUniprom-ChrB, pUniprom-ChrBopt, and pSB1C3-pChr-GFP into E. coli JM110 chassis. Setting up a backup restriction digest of pSB1C3 with EcoRI and SpeI in case the double ligation pSB1C3-pChr-GFP is unsuccessful.

Protocols Used: Transformations, Restriction digest

Results: Figure 11

Next Steps: Purification of recombinant vectors and confirmation of size and sequence. Store digested pSB1C3 at -20^oC.

Aim of experiment: Purification of recombinant vectors, pUniprom-ChrB, pUniprom-ChrBopt, and pSB1C3-pChr-GFP, for size and sequence confirmation. Patch plating of the same colonies for storage and retrieval once size and sequence have been confirmed.

Protocols Used: Overnight cultures, Patch plating

Results: N/A

Next Steps: Purification of the recombinant plasmids for confirmation of size and sequence.

Aim of experiment: Purification of pUniprom-ChrB, pUniprom-ChrBopt, and pSB1C3-pChr-GFP from overnight for confirmation of size and sequence. Restriction digest of pUnipromChrB and optimised pUniprom-ChrB optimised with BamHI and HindIII and of pSB1C3-pChr-GFP with EcoRI/PstI for size confirmation.

Protocols Used: Miniprep, Restriction digest, Colony PCR, Sequencing

Results:

Figure 12 Size confirmation of ChrB and ChrBopt.

Next Steps: Confirmation of sequence of ChrB-opt.

Aim of experiment: Set up overnight cultures of ChrBopt colonies 2, 4, 9, and 12 for sequencing.

Protocols Used: Overnight cultures

Results: N/A

Next Steps: Purification of recombinant plasmids.

Aim of experiment: To purify recombinant plasmids for sequence confirmation.

Protocols Used: Miniprep, Sequencing

Results: ChrBopt showed HindIII restriction sites and was hence cleaved into partial inserts.

Next Steps: Use alternative cloning protocol for ChrB and ChrBopt.

Aim of experiment:To compare colonies on the patch plate of pSB1C3-pChr-GFP by differentially visualising colonies that do or do not express GFP under UV light. The background of this experiment was that if there is one colony that has both inserts in the correct orientation with respect to each other, then there is perhaps one that also has them with the correct orientation with respect to the plasmid. Colonies identified as expressing GFP were patch plated and grown in liquid cultures over night in order to confirm the sequence of the insert.

Protocols Used: Patch-plating, overnight cultures

Results: Figure 15 pSB1C3-pChr-GFP in E. coli JM110.

Next Steps: Sequence confirmation of newly selected colonies.

Aim of experiment:Purification of newly selected colonies, 1, 5, 6, and 7, containing pSB1C3-pChr-GFP for subsequent sequence confirmation. Restriction digest of pUniprom with BamHI and PstI to employ an alternative cloning strategy of ChrB and ChrBopt.

Protocols Used:

Miniprep and Sequencing of pChrGFP insert.

Restriction digest of pUniprom.

Results:

Figure 16 Alignment of sequencing results.

Figure 17 Gel after restriction digest of pUniprom.

Next Steps:Stepwise ligation of pChr and GFP into pSB1C3 in order to clone the inserts in the correct orientation with respect to the plasmid. Insertion of ChrB and ChrBopt into pUniprom via BamHI and PstI links.

Aim of experiment: Ligation of pChr into pSB1C3 via EcoRI SpeI link. This is the first step of a 2-step cloning strategy with the aim of cloning pChr and GFP into pSB1C3 in the correct orientation with respect to the plasmid.

Protocols Used: Ligation, Transformation

Results: Figure 18 Plates after transformation of pChr into pSB1C3.

Next Steps: Sequence confirmation of pChr in pSB1C3.

Aim of experiment: Set up overnight cultures of pSB1C3-pChr colonies to prepare pSB1C3-pChr for sequence confirmation

Protocols Used: Overnight culture, Patch-plating

Results: N/A

Next Steps: Purification of the recombinant vector for size and sequence confirmation.

Aim of experiment: Purification of pSB1C3-pChr for sequence confirmation before ligating GFP into it.

Protocols Used:

Plasmid purification, Sequencing

Results: Figure 19 Alignment of sequenced pSB1C3-pChr vectors with pChr sequence.

Next Steps: Digest of pSB1C3-pChr with SpeI/PstI and ligation of GFPmut2.

Aim of experiment: Amplification of ChrB and ChrBopt for insertion into pUniprom. Restriction digest of the PCR product with BamHI and PstI for producing compatible sticky ends

Protocols Used: PCR-amplification, Restriction digest

Results: N/A

Next Steps: Ligation and Transformation of ChrB and ChrBopt.

Aim of experiment: Insertion of ChrB and optimised ChrB into pUniprom vector.

Protocols Used: Ligation

Results: N/A

Next Steps: Transformation of ligated vectors.

Aim of experiment:Transformation of the repressors, ChrB and ChrBopt, into JM110

Protocols Used: Transformation

Results: Figure 20 Plates after transformation.

Next Steps: Purification of pUniprom-ChrB and pUniprom-ChrBopt for sequence confirmation.

Aim of experiment: Digesting sequence confirmed pSB1C3-pChr with SpeI and PstI for subsequent ligation with GFP. Overnight cultures of ChrB and ChrBopt for subsequent plasmid purification, size confirmation, and sequence confirmation.

Protocols Used:

Restriction digest, Ligation of GFP into pSB1C3-pChr.

Overnight culture of of ChrB and ChrBopt.

Next Steps: Transformation of recombinant vector for confirmation of size and sequence.

Results: N/A

Next Steps: Transformation of pSB1C3-pChr-GFP for size and sequence confirmation. Purification of ChrB and ChrBopt for sequence confirmation.

Aim of experiment: Purification of ChrB and ChrBopt for confirmation of size and sequence.

Protocols Used: Plasmid purification, Restriction digest, Sequencing

Results:

Figure 21 Confirmation of insert size.

Figure 22 Confirmation of sequence.

Next Steps: Confirmation of expression of ChrB and ChrBopt by western blotting.

Aim of experiment: Digest of pSB1C3-pChr with SpeI and PstI for insertion of GFP. Repeat of unsuccessful transformation from 28/07.

Protocols Used: Restriction digest, Ligation, Transformation

Results: Figure 23 Plates after transformation

Next Steps: Overnight cultures for purification of the pSB1C3-pChr-GFP for confirmation of insert size and sequence.

Aim of experiment: Overnight cultures for subsequent plasmid purification of pSB1C3-pChr-GFP.

Protocols Used: Overnight culture

Results: N/A

Next Steps: Purification of the Plasmids for confirmation of insert size and sequence.

Aim of experiment: Purification of pSB1C3-pChr-GFP for confirmation of insert size and sequence.

Protocols Used: Plasmid purification, Restriction digest, Sequencing

Results:

Figure 24 Gel for size confirmation.

Figure 25 Sequence confirmation.

Next Steps: Overnight cultures in preparation for western blotting for characterisation of expression of both, GFP, and ChrB and ChrBopt.

Aim of experiment: Overnight cultures of sequence confirmed ChrB, ChrBopt, pChrGFP1 (from 15/07), pChrGFP3 and pChrGFP4 (31/07) for subsequent western blotting.

Protocols Used: Overnight culture

Results: N/A

Next Steps: Western blotting for characterisation of expression of both, GFP, and ChrB and ChrBopt.

Aim of experiment: To determine whether or not the required proteins, GFP, and ChrB, are expressed by blotting against GFP and against a 6His-tag respectively.

Protocols Used: SDS-PAGE and western blot

Results: This procedure was repeated several times during this week in order to find appropriate concentrations and volumes to load. Figure 26 was blotted after SDS-PAGE with the following concentrations:

• GFP: Dilute pellet of 1ml overnight culture with 1ml TBS. Take 100µl of this solution and mix with 100µl Laemmli Buffer. Load 3µl on the SDS-gel.
• HIS: Dilute pellet of 1ml overnight culture with 200µl Laemmli buffer. Load 10µl on the SDS-gel.

Figure 26

Next Steps:Transformation of both plasmids into MG1655 with the goal to characterise the interaction between ChrB and pChr. Furthermore the original biobrick, BBa_K1058008 will be tested under the same conditions for comparison.

Aim of experiment: To transform the following combinations of pSB1C3-pChr-GFP and pUniprom-ChrB into MG1655:

• pUniprom-ChrB2 (29/07) - pSB1C3-pChr-GFP (31/07)
• pUniprom-ChrB2 (29/07) - pSB1C3-pChr-GFP (15/07)
• pUniprom-ChrBopt3 (29/07) - pSB1C3-pChr-GFP (31/07)
• pUniprom-ChrBopt3 (29/07) - pSB1C3-pChr-GFP (15/07)

Protocols Used: Plasmid purification, Transformation

Results: Figure 27

Next Steps: Making newly transformed cells competent again in order to transform the second plasmid as well.

Aim of experiment: To prepare overnight cultures of yesterdays transformaions in order to make them competent to transform the second plasmid as well.

Protocols Used: Overnight culture

Results: N/A

Next Steps: Make cells of overnight culture competent to transform in second plasmid.

Aim of experiment:To make transformed cells competent in order to transform the second plasmid. The same plasmids that were used for the initial transformations were also used for this set of transformations.

Protocols Used: Competent cells, Transformations

Results: N/A

Next Steps: Blot against GFP in order to confirm (or refute) that ChrB interacts with pChr and hence switches off expression of GFP.

Aim of experiment: To build a strain of E. coli MG1655, containing both plasmids required for a chromate sensor.

Protocols Used: Transformation

Results: No successful transformants were found.

Next Steps: Repeat this experiment but transform pSB1C3-pChr-GFP (15/07) and pSB1C3-pChr-GFP (31/07) into competent MG1655+pUniprom-ChrB2 and MG1655+pUniprom-ChrBopt3 respectively.

Aim of experiment: To build a strain of MG1655, containing both plasmids required for a chromate sensor.

Protocols Used: Transformation

Results: Successful transformants were found on all plates.

Next Steps: To determine expression of ChrB and ChrBopt (via 6His-tag) and expression of GFP.

Aim of experiment: To determine whether ChrB, ChrBopt, and GFP are expressed and have first estimates of differences in expression levels.

Protocols Used: Overnight culture

Results: N/A

Next Steps: Lyse cells and prepare samples for SDS-PAGE + Western-Blot.

Aim of experiment: To determine whether expression of ChrB or ChrBopt has an effect on the expression of GFP.

Protocols Used: SDS-PAGE + Western Blot

Results: Figure 28

Next Steps: Check if different chromate salts have an effect on the respective expression levels.

Aim of experiment: To design a series of plate reader experiments to measure OD₆₀₀ and relative fluorescence of the chromate sensor as a response to different concentrations of K₂CrO₄, K₂Cr₂O₇, CrCl₃, and K₂SO₄.

Protocols Used: Plate Reader Experiments

Results: Figure 29 MG1655 + BBa_K1058008

Results: Figure 30 MG1655 + pSB1C3-pChr-GFP + pUniprom-ChrB

Results: Figure 31 MG1655 + pSB1C3-pChr-GFP + pUniprom-ChrBopt

Results: Figure 32 MG1655 + pSB1C3-pChr-GFP

Next Steps: Try the system with different expression levels of ChrB and ChrBopt. Induce overexpression by exploiting the T7-promoter in pUniprom after transforming the different combinations between pSB1C3-pChr-GFP and pUniprom-ChrB/pUniprom-ChrBopt into BL21(DE3) and adding IPTG.

Aim of experiment: The high-copy plasmid arrived at a concentration of 7 ^ng/_µl. Hence it was transformed in order to produce more of it before purifying and digesting it.

Protocols Used: Transformation,

Overnight culture for new E. coli MG1655

Results: No transformants were found.

Next Steps: Retry transformation with new competent E. coli MG1655.

Aim of experiment: Test transformation of BBa_K1058008 into new MG1655 competent cells and re-transformation of pSB1K3 for subsequent miniprep.

Protocols Used: Transformation

Results: Successful test transformation, but no colonies on pSB1K3 plate.

Next Steps: Use an alternative compatible plasmid for ligation of codon optimised ChrB (Bielefeld).

Aim of experiment: Restriction digest of pT7KS with EcoRI and PstI as alternative to pSB1K3. Ligation of digested ChrB (Bielefeld) and transformation of pT7KS-ChrB. Also transformations of Chromate-sensor plasmids into BL21 (DE3) in the following combinations:

• pSB1C3-pChr-GFP + pUniprom-ChrB
• pSB1C3-pChr-GFP + pUniprom-ChrBopt
• pUniprom-ChrB
• pUniprom-ChrBopt

Protocols Used: Restriction Digest, Gel Extraction, Ligation

Results:Transformations of the plasmid with pT7KS-ChrB (Bielefeld) yielded 12 colonies, but also colonies on the vector control plate. Transformations of pSB1C3-pChr-GFP + pUniprom-ChrB, pSB1C3-pChr-GFP + pUniprom-ChrBopt, pUniprom-ChrB, and pUniprom-ChrBopt into BL21(DE3) were successful.

Results:

Figure 33 pT7KS-ChrB (Bielefeld) transformations

Figure 34 Transformations into BL21(DE3)

Next Steps: Confirmation of colonies with pT7KS-ChrB by colony PCR. Preparation of recombinant BL21(DE3) for plate reader experiments.

Aim of experiment: Check if any of the colonies after ligation of ChrB (Bielefeld) into pT7KS were successful despite colonies on the control plate.

Protocols Used: Colony PCR

Results: Figure 34

Next Steps: Prepare transformed BL21(DE3) for plate reader experiments. Attempt new transformation of pT7KS-ChrB (Bielefeld).

Aim of experiment: Set up overnight cultures for subsequent plate reader experiments.

Protocols Used: Overnight cultures

Results: N/A

Next Steps: Set up plate reader, collect and analyse data. Assess the effect of induction of higher repressor-expression on the level of GFP.

Aim of experiment: To design a series of plate reader experiments to measure OD₆₀₀ and relative fluorescence of the chromate sensor. At first no chromate was added to this plate in order to assess the effect of IPTG induction.

Protocols Used: Plate reader experiments

Results: Figure 35

Next Steps:Repeat experiments with added chromate substances.

Aim of experiment: To identify whether or not there is any detectable difference in both, the level of GFP, and the level of ChrB or ChrBopt, before and after induction with IPTG.

Protocols Used: SDS-PAGE + Western blot

Results: Insert blot image here.

Next Steps: Repeat the assays of the previous weeks by subcloning ChrB, ChrBopt, and ChrB (Bielefeld) into vectors with different expression levels and see how it influences the level of GFP expression. Then add different chromate salts and also probe the system with other substances, e.g. other heavy-metal salts.