Team:Dundee/labjournal manu

LABJOURNAL

BioSpray

Our forensic toolkit aims to use synthetic biology approaches to improve on the current methods used by crime scene investigators.

Chromium Detector

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Fingerprint Aging

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Week Beginning 22/06/2015

Summary

This week was all about getting started with the newly arrived biobrick BBa_K1058008

Day 1

Aim of experiment: To prepare BBa_K1058008 for sequence confirmation, storage, and further processing.

Protocols Used:

Click here to see our plating protocol.

Click here to see our overnight culture protocol.

Results: N/A

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

Day 2

Aim of experiment: To purify BBa_K1058008 for sequence confirmation.

Protocols Used:

Click here to see our miniprep protocol.

Click here to see our sequencing protocol.

Results Sequencing: Figure 1

Next Steps: PCR of parts of BBa_K1058008.

Week Beginning 29/06

Summary

During this week, BBa_K1058008 was disassembled into its constituent parts, pChr, and ChrB.

Day 1

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:

Click here to see our PCR protocol.

Click here to see our gel extraction protocol.

Click here to see our restriction digest protocol.

Results:

Figure 2: Agarose gel after PCR of pChr and ChrB.

As can be seen on the image of the gel, PCR of ChrB was not successful. pChr was further processed by restriction digest with EcoRI and PstI

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

Week Beginning 06/07

Summary

During this week ligations of pChr and ChrB into pSB1C3 were finalised and the constituent parts of our newly designed chromate sensing system were prepared for ligation and transformation.

Day 1

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 E. coli.

Overnight culture of strain, containing GFPmut2, as a fluorescent reporter for our Chromate sensing system

Protocols Used:

Click here to see our PCR protocol.

The annealing temperature was lowered in order to increase the chances of accomodating a primer that is not 100% complementary. Furthermore different concentrations of DMSO were tested.

Click here to see our gel extraction protocol.

Click here to see our restriction digest protocol.

Click here to see our ligation protocol. Ligations were made in 2:1 and 3:1 ratio.

Click here to see our Overnight culture protocol.

Results:

Figure 3: Agarose gel after PCR of ChrB.

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.

pChr-pSB1C3, ChrBs-pSB1C3, and ChrBw-pSB1C3 were transformed into E. coli MC1061.

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

Day 2

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

Protocols Used:

Miniprep for plasmid purification of overnight culture of GFPmut2.

PCR for amplification of GFPmut2 and ChrB.

Gel extraction of PCR product pf GFPmut2.

Overnight culture of MC1061 containing pChr-pSB1C3, ChrBs-pSB1C3, and ChrBw-pSB1C3 respectively. Furthermore of a strain containing the vector pUniprom.

Results: Figure 5 Since amplification of ChrB was unsuccessful, it will be repeated.

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

Day 3

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 of pChr-pSB1C3, ChrBs-pSB1C3, ChrBw-pSB1C3 and pUniprom.

PCR for amplification of pChr and ChrB.

Restriction digest for confirmation of size of pChr and ChrB.

Results: Figure 6 Agarose gel for size confirmation.

Protocols Used:

Sequencing of pChr and ChrB that showed expectes sizes.

Results: Figure 7 Confirmed sequence of pChr1.

Results: Figure 8 Confirmed sequence of ChrBw4.

Protocols Used:

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

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

Day 4

Aim of experiment: To prepare pUniprom for insertion of ChrB.

Protocols Used:

Restriction digest of pUniprom with BamHI and HindIII.

Gel extraction of digested pUniprom

Results: Figure 9

Next Steps: Ligation of ChrB into pUniprom.

Aim of experiment: To amplify ChrB version with first 15 codons optimised for insertion into pUniprom.

Protocols Used:

PCR of optimised ChrB from the sequence confirmed ChrBw4-pSB1C3, adding restriction sites compatible to pUniprom.

Gel extraction of optimised ChrBafter PCR.

Results: Figure 10

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

Day 5

Aim of experiment: Ligation of ChrB and optimised ChrB into pUniprom. Ligation of pChr and GFPmut2 into pSB1C3

Protocols Used:

Ligation of ChrB and optimised ChrB into pUniprom via BamHI and HindIII restriction sites. Ligation of pChr to GFPmut2 via Spe/Xba link and ligation of pChrGFP construct into pSB1C3 EcoRI/PstI restriction sites.

Results: N/A

Next Steps:Transformation of abovementioned ligations.

Week Beginning 13/07

Summary

During this week successful transformations were identified and propagated.

Day 1

Aim of experiment: Transform recombinant vectors into E. coli chassis.

Protocols Used:

Transformation of ChrB-pUniprom, optimised ChrB-pUniprom and pChr-GFPmut2-pSB1C3 into JM110.

Results: Figure 11

Aim of experiment: Backup of digested pSB1C3 for the case that a ligation with 2 inserts does not work.

Protocols Used:

Restriction digest of pSB1C3 with EcoRI/SpeI for subsequent insertion of pChr.

Results:N/A

Next Steps: Store digested plasmid at -20^oC.

Day 2

Aim of experiment: Purification of recombinant vectors for sequence and size confirmation.

Protocols Used:

Overnight culture of JM110 with ChrB-pUniprom, optimised ChrB-pUniprom and pChr-GFPmut2-pSB1C3.

Patch plating of the same colonies.

Results: N/A

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

Day 3

Aim of experiment: Purification of recombinant plasmids for confirmation of size and sequence.

Protocols Used:

Miniprep of ChrB-pUniprom, optimised ChrB-pUniprom and pChr-GFPmut2-pSB1C3.

Results:N/A

Protocols Used:

Restriction digest of ChrB-pUniprom and optimised ChrB-pUniprom with BamHI and HindIII. Restriction digest of pChr-GFPmut2-pSB1C3 with EcoRI/PstI.

Results: Figure 1

Protocols Used:

Colony PCR of pUniprom-ChrB-opt for elucidation of insert size or presence of insert.

Results: Figure 13 Gel after colony PCR. Colonies 2, 4, 9, and 12 were selected for sequencing.

Protocols Used:

Sequencing of pSB1C3-pChr-GFP.

Results: Figure 14 Sequencing result of pSB1C3-pChr-GFPmut2_1. The Result for pSB1C3-pChr-GFPmut2_2 was not as expected, albeit in the correct orientation.

Next Steps: Confirmation of insert size and sequence of ChrB-opt.

Day 4

Aim of experiment: To prepare ChrB-opt colonies 2, 4, 9, and 12 for sequencing.

Protocols Used:

Click here to see our overnight culture protocol.

Results:N/A

Next Steps: Purification of recombinant plasmids.

Day 5

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

Protocols Used:

Miniprep of ChrB-opt 2, 4, 9, 12.

Results:N/A

Protocols Used:

Sequencing of ChrB-opt 2, 4, 9, 12.

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

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

Week Beginning 20/07

Summary

During this week an alternative cloning strategy for ChrB and ChrB-opt was employed. Furthermore, the pChr-GFP construct was cloned in a stepwise fashion.

Day 1 - 20/07

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.

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

Next Steps:Size confirmation and sequencing of newly selected colonies.

Day 2 - 21/07

Aim of experiment:Purification of newly selected colonies containing pSB1C3-pChr-GFP for subsequent sequence confirmation.

Protocols Used:

Miniprep for purification of pSB1C3-pChr-GFP.

Results:N/A

Next Steps:Sequencing of pSB1C3-pChr-GFP samples 1, 5, 6, and 7.

Protocols Used:

Sequencing of pSB1C3-pChr-GFP samples 1, 5, 6, and 7.

Results: Figure 16 Alignment of sequencing results.

Next Steps:Stepwise ligation of pChr-GFP into pSB1C3.

Protocols Used:

Restriction digest of pUniprom with BamHI/PstI for subsequent insertion of ChrB and ChrB-opt.

p>Results: Figure 17 Gel image

Next Steps:Insertion of ChrB and ChrB-opt respectively via BamHI/PstI.

Day 3 - 22/07

Aim of experiment:Ligation of pChr into pSB1C3 via Eco/Spe link.

Protocols Used:

Ligation of pChr(08/07/15) into pSB1C3(13/07/15)

Results: N/A

Aim of experiment:Transformation

Protocols Used:

Transformation of pSB1C3-pChr into JM110.

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

Next Steps:Sequence confirmation of pChr in pSB1C3.

Day 4 - 23/07

Aim of experiment: To prepare pSB1C3-pChr for sequence confirmation

Protocols Used:

Overnight culture of pSB1C3-pChr.

Patch-plating of the same colonies of pSB1C3-pChr.

Results: N/A

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

Day 5 - 24/07

Aim of experiment:Purification of pSB1C3-pChr for sequence confirmation.

Protocols Used:

Miniprep of pSB1C3-pChr.

Results: Insert table here

Aim of experiment:Sequence confirmation of pSB1C3-pChr.

Protocols Used:

Sequencing of samples 1 and 2 of pSB1C3-pChr.

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.

Day 6 - 25/07

Aim of experiment: Amplification of ChrB and optimised ChrB

Protocols Used:

PCR-mplification of ChrB and ChrBopt.

Results: N/A

Next Steps:Ligation and Transformation of ChrB and optimised ChrB.

Day 7 - 26/07

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

Protocols Used:

Ligation of ChrB and optimised ChrB into vector pUniprom.

Results: N/A

Next Steps: Transformation of ligated vectors.

Week Beginning 27/07

Summary

Final touches were made on the chromate system. All parts were ligated into their final vectors during this week. Preparations were made for characterisation of each of the parts.

Day 1 - 27/07

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

Protocols Used:

Transformation of ChrB and optimised ChrB into JM110

Results: Figure 1

Next Steps: Purification of the plasmids for sequence confirmation.

Day 2 - 28/07

Aim of experiment:Processing of pSB1C3-pChr from 24/07 for insertion of GFP.

Protocols Used:

Restriction digest of pSB1C3-pChr with SpeI/PstI for insertion of GFP.

Ligation of GFPmut2, previously digested with XbaI/PstI.

Results: N/A

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

Aim of experiment:Overnight culture from plates of ChrB and optimised ChrB.

Protocols Used:

Overnight culture of of ChrB and optimised ChrB.

Results: N/A

Next Steps: Purification of plasmids for sequence confirmation.

Day 3 - 29/07

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

Protocols Used:

Miniprep of ChrB and ChrBopt.

Restriction Digest of the purified vectors for confirmation of size.

Sequencing of ChrB and ChrBopt.

Results:

Confirmation of insert size.

Confirmation of sequence.

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

Day 4 - 30/07

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

Protocols Used:

Restriction digest of pSB1C3-pChr with SpeI/PstI for insertion of GFP.

Ligation of GFPmut2, previously digested with XbaI/PstI.

Transformation of pChrGFP into JM110.

Results: Figure 22

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

Day 5 - 31/07

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

Protocols Used:

Overnight cultureof pSB1C3-pChr-GFP.

Results: N/A

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

Day 6 - 01/08

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

Protocols Used:

Miniprep for purification of pSB1C3-pChr-GFP.

Restriction digest of purified plasmid with EcoRI/PstI for confirmation of insert size.

Results:

Figure 24 Gel for size confirmation.

Figure 24 Sequence confirmation.

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

Day 7 - 02/08

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 of named constituents.

Results: N/A

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

Week Beginning 03/08

Summary

This week was entirely used for characterising the expression of GFP from the promoter pChr, and the expression of ChrB from the tat-promoter in pUniprom.

Day 1 - Day 7

Aim of experiment:To determine whether or not the required proteins, GFP, and ChrB, are expressed.

Protocols Used:

SDS-PAGE and western blot against GFP and against His.

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.

Week Beginning 10/08

Summary

This week was spent with transforming all combinations of plasmids into MG1655. Tests for their interaction were also prepared.

Day 1 - 10/08

Aim of experiment:To transform the following combinations of plasmids into MG1655:

• ChrB2 (29/07) - pChrGFP (31/07)
• ChrB2 (29/07) - pChrGFP (15/07)
• ChrBopt3 (29/07) - pChrGFP (31/07)
• ChrBopt3 (29/07) - pChrGFP (15/07)

in a stepwise fashion.

Protocols Used:

Miniprep all 4 constituents. Also purify BBa_K1058008

Transform all 4 in separate aliquots of MG1655.

Results: Figure 1

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

Day 2 - 11/08

Aim of experiment: To prepare overnight cultures of the transformations in order to make them competent again.

Protocols Used:

Overnight culture of a colony of each plate.

Results: N/A

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

Day 3 - 12/08: Made competent MG1655 with ChrB2, ChrBopt3, pChrGFP (15/07), pChrGFP (31/07).

Aim of experiment:To make transformed cells competent in order to transform the second plasmid for a full system.

Protocols Used:

Making competent cells of MG1655, containing ChrB2, ChrBopt3, pChrGFPrev, pChrGFPfor respectively.

Transform same plasmids that were used for initial transformation into newly made competent cells in order to get all 4 combinations of plasmids as outline on day 1 of this week.

Results: insert images of plates once they are round.

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

Day 4 - 13/08: Transformed ChrB2 and ChrBopt3 into MG1655+pChrGFP(15/07) and MG1655+pChrGFP(31/07)

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

Protocols Used: Transformation

Results: No successful transformants were found.

Next Steps: Check Plates for successful transformants.

Week Beginning 17/08

Summary

During this week recombinant MG1655 were made, containing both plasmids. Western Blots were used to determine expression of ChrB, ChrBopt, and GFP.

Day 1 - 17/08: Transformations akin to 13/08 were done, but pChrGFP(15/07) and pChrGFP(31/07) were transformed into MG1655+ChrB2 and MG1655+ChrBopt3.

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 His-tag) and expression of GFP.

Day 2 - 18/08: Preparation of samples for western blot.

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.

Day 3-6 - 19/08 - 22/08: Repeated western blots with different concentrations to determine whether the expression of ChrB has an effect on the expression of GFP.

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

Protocols Used: SDS-PAGE + Western Blot

Results: Figure 28

Next Steps: Check if different substances containing chromate have an effect on the expression.

Week Beginning 24/08

Summary

Plate reader experiments were carried out in order to determine the effect of K₂CrO₄, K₂Cr₂O₇, CrCl₃, and K₂SO₄ on the expression of GFP.

Day 1 - Day 7 - 24/08-30/08: Design of Plate reader experiments.

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

Results: Figure 30

Results: Figure 31

Results: Figure 32

Next Steps: Try the system with different expression levels of repressor. Induce overexpression of repressor from T7-promoter in pUniprom using BL21 and IPTG.

Week Beginning 31/08

Summary

UK Synbio Conference and UK iGEM meetup at Westminster University.

Week Beginning 07/09

Summary

Parts from CeBiTec Bielefeld arrived and attempts at cloning were made. The chromate sensing system was transformed into BL21 and some of the plate reader experiments repeated.

Day 1 - 07/09: Transformation of pSB1K3 into MG1655.

Aim of experiment: The high-copy plasmid arrived at very low concentration. Hence it was transformed in order to produce more of it before digesting it.

Protocols Used: Transformation, Overnight culture for competent cells

Results: No transformants were found.

Next Steps: Retry and make more competent MG1655.

Day 2 - 08/09: Transformation of pSB1K3 into MG1655.

Aim of experiment: Test transformation of BBa_K1058008 into new MG1655 competent cells and transformation of pSB1K3-plasmid

Protocols Used: Transformation,

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

Next Steps: Use an alternative plasmid.

Day 3 - 09/09: Transformation of pSB1K3 into MG1655.

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

• pChrGFP + ChrB
• pChrGFP + ChrBopt
• ChrB
• ChrBopt

Protocols Used: Restriction Digest, Gel Extraction, Ligation

Results:Transformations of the plasmid with ChrB (Bielefeld) yielded 12 colonies, but also colonies on the vector control plate. Transformations into BL21 were successful.

Results: Figure 33 Figure 34

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

Day 4 - 10/09: Colony PCR of pT7KS.

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

Protocols Used: Colony PCR

Results: Figure 34

Next Steps: Prepare recombinant BL21 for plate reader experiments. Attempt new transformation of ChrB (Bielefeld).

Day 5 - 11/09: Preparation for Plate reader experiment with BL21 (DE3)

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.

Day 6 - 12/09: Set up plate reader.

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

Results:add images once analysed.

Next Steps:Repeat experiments with added chromate substances.

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Plate Reader Protocol

1. A 5ml overnight culture of the desired strain was grown at 37^oC.
2. Equalise OD₆₀₀ if required. Dilute culture 1:1000.
3. Aliquot 198µl into the required amount of wells in a 96-well plate.
4. Add additional substances and controls as required.
5. Preheat platereader to required temperature and start experiment.
6. Retrieve and analyse data.

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Competent Cells Protocol

1. A 5ml overnight culture of the desired strain was grown at 37^oC.
2. Subsample ¹/₁₀ of the volume of the subculture. I.e. 1ml of the overnight culture into 10ml fresh LB.
3. Grow subculture at at 37^oC for 2h.
4. Spin down subculture at 4000rpm at 4^oC for 10min.
5. Resuspend the pellet in ice cold transformation buffer. Use ¹/₁₀ of the initial volume of the subculture.
6. Use 100µl per transformation.

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Plating Protocol

1. Agar plates with required antibiotic were taken out of 4^oC to warm to room temperature.
2. Cells or colonies were picked up from the source with a toothpick under sterile conditions.
3. Fresh agar plates were inoculated with cells on the toothpick.
4. Agar plates were incubated at 4^oC over night.

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Sequencing Protocol

For sequencing a sample, the following ingredients were added to a microcentrifuge tube:

• Volume of sample equivalent to 550ng DNA
• 2µl of 3mM primer
• Remaining volume of water for a total volume of 30µl

The samples were submitted to DNA sequencing and services.

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Overnight Culture Protocol

1. Agar plates with required antibiotic were taken out of 4^oC to warm to room temperature.
2. Cells or colonies were picked up from the source with a toothpick under sterile conditions.
3. 5ml of LB medium, supplemented with the required antibiotic, were inoculated with the toothpick.
4. Tubes were incubated at 37^oC in a rotary incubator at 200rpm over night.

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PCR Protocol

PCR reaction mixtures were set up according to the table below. The length of all sequences used is stated in the Sequences section.

Reactants	Volume (µl)
DNA	1
DMSO	2.5
Forward Primer	0.5
Reverse Primer	0.5
Herculase Buffer	10
dNTP	0.5
Herculase	0.5
H2O	34.5

**PCR Programme**

Elongation time was altered according to the length of the sequences.

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Colony PCR Protocol

The PCR reaction mixture were set up in multiples of the volumes in table below. The length of all sequences used is stated in the Sequences section.

The DNA template is made in the following way:

1. Agar plates with required antibiotic were taken out of 4^oC to warm to room temperature.
2. A numbered grid according to the desired number of PCR reactions is drawn on the back of the plate.
3. Cells or colonies were picked up from the source with a toothpick under sterile conditions.
4. Each colony is transferred to a 30µl aliquot of sterile water.
5. With the same toothpick the grid with the respctive number on the plate was inoculated.
6. • The plate was incubated at 37^oC overnight.
   • The inoculated water-aliquots were boiled for 10min, then centrifuged at 13300rpm for 10min.
7. The supernatant of this preparation is the DNA fraction for the subsequent PCR reaction.

Reactants	Volume (µl)
DNA	5
DMSO	1
Forward Primer	0.5
Reverse Primer	0.5
+Mg Buffer	2
dNTP	0.2
Taq-polymerase	0.2
H2O	10.6

15µl of the reaction mixture was aliquoted into separate PCR tubes. 5µl of DNA fraction was added to each respective tube in order to have a total reaction volume of 20µl per tube.

**PCR Programme**

Elongation time was altered according to the length of the sequences.

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Miniprep Protocol

1. 5ml bacterial overnight culture was pelleted by stepwise centrifugation in 1.5ml microcentrifuge tubes at 13300rpm for 3 minutes at room temperature.
2. Pelleted bacterial cells were resuspended in 250µl of Buffer P1.
3. 250µl of Buffer P2 was added and mixed thoroughly by inverting the tube 4-6 times until solution became clear.
4. 350µl of Buffer N3 was added and immediately and thoroughly mixed by inverting the tube 4-6 times.
5. The mixture was centrifuged for 10 minutes at 13300rpm in a microcentrifuge.
6. The supernatant was applied to a QIAprep spin column by pipetting.
7. 500µl of Buffer PB was added to the QIAprep spin column and centrifuged for 1 minute. The flow-through was discarded.
8. 750µl of Buffer PE was added to the QIAprep spin column and centrifuged for 1 minute. The flow-through was discarded.
9. The QIAprep spin column was spun for an additional minute to remove residual wash buffer.
10. The QIAprep spin column was placed in a clean 1.5ml microcentrifuge and the DNA eluted by adding 30µl of H₂O to the QIAprep spin column and centrifuging for 1 minute.

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Gel Extraction Protocol

1. The desired DNA fragment was excised from the agarose gel with a clean, sharp scalpel.
2. The gel slice was transferred into a microcentrifuge tube. 700µl of Buffer QG was added.
3. The gel slice was incubated in a water bath at 50°C for 10 min (or until the gel slice has completely dissolved). The tube was vortexed every 2–3 min to help dissolve gel.
4. After the gel slice had been dissolved completely, the color of the mixture was checked that it was yellow (similar to Buffer QG without dissolved agarose). If the color of the mixture was orange or violet, 10 μl 3 M sodium acetate, pH 5.0 would be added, and mixed. The color of the mixture would turn yellow.
5. 230µl of isopropanol was added to the sample and mixed.
6. A QIAquick spin column was placed in a 2ml collection tube. The sample was applied to the QIAquick spin column and centrifuged for 1 minute. The flow-through was discarded.
7. 500µl of QG buffer was added to the QIAquick spin column and centrifuged for 1 minute. The flow-through was discarded.
8. 750µl of Buffer PE was added to the QIAquick spin column and centrifuged for 1 minute. The flow-through was discarded.
9. The QIAprep spin column was spun for an additional minute to remove residual wash buffer.
10. The QIAprep spin column was placed in a clean 1.5ml microcentrifuge and the DNA eluted by adding 30µl of H2O to the QIAprep spin column and centrifuging for 1 minute.

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Restriction Digest Protocol

Restriction digests were set up according to the table below. The appropriate reaction and restriction enzymes that were used is stated in the Journal section.

	Post Plasmid Purification	Post PCR	gBlock
DNA	1mg	50µl	100ng
Cutsmart Buffer (µl)	6	6	6
Water (µl)	As required	1	As required
Enzyme 1 (µl)	1.5	1.5	1.5
Enzyme 2 (µl)	1.5	1.5	1.5
Total Volume (µl)	30	60	30

• The digests were incubated at 37^oC for 3 hours.
• Note that when performing plasmid digestions, 2.5µl of alkaline phosphatase and 2.5µl of its buffer were added at the 2 hour and 2.5 hour mark.

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Ligation Protocol

Ligation reactions were set up according to the table below. The vector to insert ratio is stated for each specific reaction in the Journal section.

	Vector (µl)	2:1 (Vector: Insert) [µl]	3:1 (Vector: Insert) [µl]	4:1 (Vector: Insert) [µl]
T4 DNA Ligase	1	1	1	1
Ligase Buffer	1	1	1	1
Vector	1	1	1	1
Insert	-	2	3	4
H20	7	5	4	3

The reactions were incubated at room temperature for at least 3 hours.

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Transformation Protocol

1. 100µl of competent cells were defrosted. Thaw on ice.
2. Agar plates were taken out of 4^oC to warm to room temperature.
3. 1-5µl of DNA was added to cells.
4. Cell and DNA mixture was left on ice for 20 minutes.
5. Mixture was placed in 42^oC water bath for 90 seconds.
6. Mixture was put back on ice for 2 minutes.
7. 1ml of LB (without antibiotic) was added to cells.
8. Cells were allowed to grow for 1 hour at 37^oC in a shaking incubator.
9. Cells were then spun down and the supernatant discarded.
10. The pellet was resuspended in the 100µl.
11. Cells were plated on agar plates with appropriate antibiotic.

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Protein Expression Optimization Protocol

SDS-PAGE and Western Blotting

1. 50µl of an overnight culture was used to inoculate 5ml of fresh LB containing the appropriate antibiotics and left to grow at 37^oC in a shaking incubator.
2. Once the OD₆₀₀=0.6-0.8, 1mM of IPTG was added to the culture and left to grow for a further 3 hours.
3. 1ml of each culture was taken and spun down in a microcentrifuge for 3 minutes.
4. Meanwhile, 950µl of 2x Laemmli sample buffer was added to 50µl of B-mercaptoethanol.
5. The supernatant was discarded from the samples and the pellet resuspended in 200µl of the above solution.
6. The samples were then boiled at 90^oC for 10 minutes.
7. Afterwards, they were spun down for 1 minute in a microcentrifuge.
8. At this stage, the samples could be stored at -20^oC or ran on a gel immediately.
9. Two gradient gels consisting of a 12% separating gel and a stacking gel were prepared and the samples ran at 100V until proteins reach end of the stacking gel at which point the voltage was increased to 200V for 20 minutes.
10. One gel was stained using Instant Blue and the other was used to transfer the proteins onto a membrane.
11. After the proteins had been transferred onto the membrane, the membrane was blocked in 5% Marvel milk overnight at 4^oC.
12. The next day, the membrane was washed 3 times in 1xTBST buffer and then suspended in 10ml of 1xTBS buffer to which the primary anti-His antibody was added and left for one hour. The protein ladder was cut off before adding the primary antibody.
13. The membrane was then washed 3 times in 1xTBST buffer for 5 minutes and then suspended in 10ml of 1xTBS buffer, to which the secondary antibody was added and left for one hour.
14. The membrane was then washed in 3x10ml 1xTBST buffer for 5 minutes.
15. The blot was then soaked in developing solution for 5 minutes before being wrapped in a plastic cover aligned with the protein ladder that was cut off at step 12.
16. Once in the developing room, the film was exposed to the blot for 30 seconds, 2 minutes and 5 minutes and then placed in the film developer.

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Figure 1: Alignment of BBa_K1058008. Sequences from top to bottom: sequencing result from forward primer; expected sequence; reverse complement of sequencing result from reverse primer; sequencing result from intermediate primer. Colour code: Green = Prefix/Suffix; Pink = Annealing site of intermediate primer; Yellow = Mismatch in one sequence; Red = In frame stop codons.

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Figure 2: 1% agarose gel after PCR of BBa_K1058008 with primers specific to the promoter region, pChr, and the repressor region, ChrB. Left: pChr (expected size 169bp); Middle: 1kb+ ladder, Right: ChrB (expected size 939bp).

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Figure 3: 1% agarose gel after PCR of BBa_K1058008 with primers specific to the repressor region, ChrB. Left: 1.5µl DMSO; Middle: 1kb+ ladder, Right: 2.5µl DMSO. Expected size for each: 939bp.

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Figure 4: 1% agarose gel after PCR of GFPmut2 with corresponding primers primers. Left: GFPmut2 (expected size 717bp); Middle: 1kb+ ladder, Right: PCR product of ChrB, using primers to add a BamHI restriction site in front of it and a His-Tag and a HindIII restriction site at the end. (expected size of ChrB: 939bp).

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Figure 5: 1% agarose gel after restriction digest of pChr-pSB1C3, ChrBs-pSB1C3, and ChrBw-pSB1C3 for confirmation of size. Ladder: 1kb+. Top: 1-4: pChr 1-4; ChrBs 1-2. Bottom: ChrBs 3-4; ChrBw 1-4. Expected sizes: pChr: 169bp; ChrB: 939bp.

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Figure 6: 1% agarose gel after PCR of GFPmut2 with corresponding primers primers. Left: GFPmut2 (expected size 717bp); Middle: 1kb+ ladder, Right: PCR product of ChrB, using primers to add a BamHI restriction site in front of it and a His-Tag and a HindIII restriction site at the end. (expected size of ChrB: 939bp). Colours: Green: Prefix and Suffix; Red: In frame stop codons.

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Figure 7: 1% agarose gel after PCR of GFPmut2 with corresponding primers primers. Left: GFPmut2 (expected size 717bp); Middle: 1kb+ ladder, Right: PCR product of ChrB, using primers to add a BamHI restriction site in front of it and a His-Tag and a HindIII restriction site at the end. (expected size of ChrB: 939bp). Colours: Green: Prefix and Suffix; Red: In frame stop codons; Blue: First 15 codons optimised for expression in E. coli.

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Figure 8: 1% agarose gel after restriction digest of pUniprom (Expected size 2454bp).

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Figure 10: 1% agarose gel after PCR of ChrBw4 (Expected size 939bp).

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Figure 11: Plates after transformation of ChrB-pUniprom, optimised ChrB-pUniprom and pChr-GFPmut2-pSB1C3 into JM110.

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Figure 12: 1% agarose gel after restriction digest ofChrB-pUniprom, optimised ChrB-pUniprom and pChr-GFPmut2-pSB1C3 for confirmation of size. Ladder: 1kb+. Top: 1-4: pSB1C3-pChr-GFP 1-4; pUniprom-ChrB-opt 1-2. Bottom: pUniprom-ChrB-opt 3-4; pUniprom-ChrB 1-4. Expected sizes: pChr-GFP: 909bp; ChrB and Chrb-opt: 939bp.

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Figure 13: 1% agarose gel after colony PCR of pUniprom-ChrB-opt. Top: colonies 1-11; Bottom: colonies 12-22. Ladder: 1kb+ Expected size: 939bp.

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Figure 14: Sequencing result of pSB1C3-pChr-GFP after double ligation. Even though the sequence is as expected, it is ligated exactly in reverse. Colours: Green: Prefix and partial Suffix; Blue: SpeI/XbaI-scar; Cyan: Ribosomal binding site, Red: In frame stop codons.

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Figure 15: Plates with pSB1C3-pChr-GFP in JM110 after double ligation und UV light. Numbered colonies were selected for initial size confirmation and sequencing. However, other colonies seemed to express GFP as well, hence a second attempt was made with different colonies from the same plate.

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Figure 16: Alignment of sequencing results of pSB1C3-pChr-GFP colonies 1, 5, 6, and 7 with GFPmut2 and pChr sequence. Colony 1 remained consistent, the other tested colonies were without success. Colours: Green: Prefix and partial Suffix; Blue: SpeI/XbaI-scar; Cyan: Ribosomal binding site, Red: In frame stop codons.

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Figure 17: 1% Agarose gel after restriction digest of pUniprom with BamHI/PstI (expected size: 2454bp). Right: PCR product of Haptoglobin.

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Figure 18: Plates after transformation of pChr into pSB1C3. Top: ratio insert:vector 2:1; Bottom: ratio insert:vector 3:1.

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Figure 19: Plates after transformation of pChr into pSB1C3. Top: ratio insert:vector 2:1; Bottom: ratio insert:vector 3:1.

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Figure 20: Plates after transformation of, Top: ChrB and into pUniprom; Bottom: optimised ChrB into pUniprom. Ratio insert:vector left: 3:1; Right: 4:1.

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Figure 21: Gel after restriction digest of Left: pUniprom-ChrB, and Right: pUniprom-ChrBopt with BamHI/PstI for confirmation of insert size. Expected size 939bp. ChrB2 and ChrBopt3 were selected for sequencing.

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Figure 22: Alignments of ChrB (left) and ChrBopt (right). Colours: Pink = Restriction sites (BamHI/PstI); Blue = Hexa-Histidine-Tag; Red = In-frame stop codons; Cyan = optimised codons.

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Figure 23: Plates after ligation of GFP into pSB1C3-pChr. Left: Ratio Insert:Vector 2:1; Right: Ration Insert:Vector 3:1.

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Figure 24: Gel for size confirmation of pSB1C3-pChr-GFP (Top). The bottom lanes are unrelated to this project. Samples 3 and 4 were submitted for sequencing.

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Figure 25: Gel for size confirmation of pSB1C3-pChr-GFP (Top). The bottom lanes are unrelated to this project. Samples 3 and 4 were submitted for sequencing.

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Figure 26: Western blot against GFP (left) and ChrB (right). Expected sizes: GFP ~ 27kDa, ChrB ~35kDa. Left: Lane1 - pChrGFP inverse from 15/07; Lane2 - pChrGFP3 normal from 31/07; Lane3 - pChr only, negative control; Lane4 - positive control. Right: Lane1 - ChrB2, Lane2 - ChrBopt3, Lane3 - pUniprom empty vector, negative control, Lane4 - positive control, expected size ~35kDa.

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Figure 27: Transformations of single constituents of Chromate sensing system.

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Figure 28: Western Blot against GFP.

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Figure 29: Results of Plate reader experiment after exposing MG1655 with BBa_K1058008 to different concentrations of chromate and control substances. One measurement was made every 10min across 16h.

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Figure 30: Results of Plate reader experiment after exposing MG1655 with pSB1C3-pChr-GFP and pUniprom-ChrB to different concentrations of chromate and control substances. One measurement was made every 10min across 16h.

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Figure 31: Results of Plate reader experiment after exposing MG1655 with pSB1C3-pChr-GFP and pUniprom-ChrBopt to different concentrations of chromate and control substances. One measurement was made every 10min across 16h.

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Figure 32: Results of Plate reader experiment after exposing MG1655 with pSB1C3-pChr-GFP to different concentrations of chromate and control substances. One measurement was made every 10min across 16h.

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Figure 33: Plates after transformation into pT7KS.

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Figure 34: Plates after transformation of chromate sensor plasmids into BL21 (DE3).

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Figure 35: Agarose gel after colony PCR.

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