Team:Macquarie Australia/Notebook

Wet lab notebook

Project planning - no wet lab work this week.

We went through lab safety induction to ensure safety procedures were followed at all times in the lab
We learnt how to perform digestion, ligation, transformation and how to make and run agarose gels
We used digestion to screen the Photosystem II (PSII) parts DCA, psbELJTB and psbMZtlwkOPQR. psbELJTB and psbMZtlwkOPQR showed successful digestion and correct sizes. DCA showed signs of contamination in all samples.
We decided to focus of the Chlorophyll plasmid rather than PS II. We proceeded with digestion and ligation of ChlD with ChlI2 as well as DRV1 with ChlG, both into Kanamycin backbones, following the 3A assembly protocol.
Renee, Areti and Karl also began the interlab study:

Made LB media + cam plates
Transformed Εscherichia coli DH5α cells with the interlab testing devices.Transformation was performed twice, due to poor colony growth in the LB + cam media at the first round of transformations.

Figure 1. Agarose gel (1%) electrophoresis of single (E) and double (E+S or E + X) digests of Photosystem II composite parts (ELJTB and MZHWKOPQR). Both ELJTB samples showed correct banding in single and double digests for the 2.1kb part (same size as backbone). 1 samples for MZHWKOPQR showed correct digestion with the 3.2kb part

We transformed the DVR1-ChlG and ChlD-ChlI2 ligation samples into competent DH5α E.coli cells. DVR1-ChlG had successful transformants which were then incubated in LB broth + Kan. ChlD-ChlI2 had given an unsuccessful transformation, so the process was repeated and was successful the second time. The samples were then liquid cultured.
Interlab experiments were completed this week:

Calibrations 1, 2 and 3 were completed early on in the week
Cell measurements (Abs600 and fluorescence) were taken as per the interlab protocol
Colony forming units per OD600 = 0.1 of the negative and positive control devices was also determined (in triplicates)

Set up backbone insertions for trc-POR, trc-ChlI1 (Digestion, ligation and transformation
Interlab experiments:

Colony forming units per OD600 = 0.1 of the negative and positive control devices was completed again.
Data analysis and submission.

The liquid cultures of DVR1-ChlG and ChlD-ChlI2 were miniprepped and digested (single, double digest) to screen for successful ligations by agarose gel electrophoresis (1% agarose).

The gel showed unsuccessful ligations so we switched our ligation technique to standard assembly and backbone swapping of necessary parts

We backbone-swapped DVR1 to Kan and were in the process of swapping ChlD, trc-POR and GUN4
Made up more plasmid stock of current parts by plasmid transformation, culturing and miniprep
Begun Standard assembly of DVR1 + ChlG

Miniprepped all samples

Begun induction and SDS-PAGE to compare lac-ChlH and trc-ChlH

Figure 2. Agarose gel (1%) electrophoresis of single (E) and double (EP) digests of lac-ChlM, lac-ChlG and lac-ChlI2. This gel was checking parts we had transformed to make up more stock of for this years project, all part sizes were correct.

Figure 3. Agarose gel (1%) electrophoresis of single (E) and double (EP) digests of plasto, ChlD, ChlP (In both Cam and Kan backbones) and POR. This gel was checking parts we had transformed to make up more stock of for this years project, Correct part sizes were observed for each part.

Ran GUN4, ChlD and trc-POR backbone swaps as well as DVR1-ChlG on a 1% agarose gel
Sent off successful samples of each for sequencing
We inserted synthesised DNA biobricks into Amp and Cam backbones for trc-POR,YidC and trc-ChlI1, trc-ycf39-HliD
We then transformed, liquid-cultured, miniprepped and screened on a 1% agarose gel
We had successes for trc-ChlI1 in Amp, which we sent for sequencing confirmation
Proceeded with standard assembly for:

CTH1-ycf54-ChlM-ter + trc-FNR-fdx or trc-FNR
trc-ChlH + GUN4
trc-POR + ChlP
ChlI2 + ChlD

We sent CTH1-ycf54-ChlM-ter-trc-FNR-fdx as well as CTH1-ycf54-ChlM-ter-trc-FNR for sequencing
Sequencing came back for trc-ChlH to show that the promoter swap was unsuccessful. A reattempt of promoter swap was then undertaken
Transformed trc-POR-ChlP and ChlI2-ChlD

Figure 4. Agarose gel (1%) electrophoresis of single (E) and double (EP) digests of trc-POR (Backbone insertion into Cam/Kan) and DVR1-ChlG ligation. Samples 2 & 3 of trc-POR (Kan) and Sample 2 of trc-POR (Cam) showed correct band size. DVR1-ChlG showed correct band size for our very first successful standard assembly (Biobrick and plasmid backbone similar size forming a large single band in double digests).

Screened trc-ChlI1 and sent 1 successful plasmid away for sequencing
Performed SDS-PAGE comparing trc-FNR-fdx and lac-FNR-fdx
Liquid-cultured, Miniprepped and screened trc-ChlH, trc-POR-ChlP, trc-ycf39-HliD, trc-FNR and trc-FNR-fdx and ChlI2-ChlD

sent ChlH, trc-POR, trc-ycf39-HliD, trc-FNR-fdx and ChlI2-ChlD plasmids for sequencing

Digestion/ligation for trc-POR+ChlP, ChlI1+ChlI2-ChlD, trc-ChlH-GUN4
Glycerol stock made for ChlH

Figure 5. Agarose gel (1%) electrophoresis of single (E) and double (EP) digests of ChlI2-ChlD and trc-POR-ChlP standard assembly ligations. All samples of ChlI2-ChlD had successful band sizes. trc-POR-ChlP did not show correct banding.

Transformed, Liquid-cultured and miniprepped trc-ChlH-GUN4, trc-ChlI1-ChlI2-ChlD and trc-POR-ChlP
Made glycerol stock for trc-POR and trc-ChlH
Digested and screened trc-ChlH-GUN4, trc-FNR and trc-FNR-fdx. Only trc-FNR-fdx and trc-ChlH-GUN4 had successes, which were sent off for sequencing
Induced trc-POR cells and ran the cell lysate on SDS-PAGE

Digested and screened trc-ChlH-GUN4, trc-ChlI1-ChlI2-ChlD and trc-POR-ChlP on a 1% agarose gel and sent successful samples for sequencing
Backbone-swapped lac-CTH1-ycf54-ChlM (Kan)
Performed standard assembly on trc-POR-ChlP-DVR1-ChlG and sent successful ones for sequencing
Induced new trc-POR samples and ran cell lysate on an SDS-PAGE
Grew barley for protochlorophyllide extraction
Made up more plasmid stock of trc-POR
Made up glycerol stock of ChlI1
Redesigned and ordered sequencing primers
Tried to insert G-block for yidC and trc-FNR into ampicillin backbone, no successes from screened samples
Digestion/ Ligation of trc-ChlI1-ChlI2-ChlD + ChlH-GUN4. Backbone swap of trc-POR (switched to Cam to send to iGEM) and lac-CTH1-ycf54-ChlM + trc-POR

Figure 6. Agarose gel (1%) electrophoresis of single (E) and double (EP) digests of trc-ChlH-GUN4, trc-POR-ChlP and trc-ChlI1-ChlI2-ChlD standar assembly ligations. trc-ChlH-GUN4 and trc-POR-ChlP showed successful ligations with correct band sizes.

Received sequencing results for trc-POR-ChlP and sent off trc-POR-ChlP-DVR1-ChlG samples for sequencing
Digested, ligated, transformed and liquid-cultured lac-CTH1-ycf54-ChlM + trc-FNR-fdx
Transformed trc-ChlI1-ChlI2-ChlD + trc-ChlH-GUN4 (shortened to trc-IH), Backbone-swapped trc-POR (to Cam to send to iGEM) and lac-CTH1-ycf54-ChlM + trc-POR

Liquid-cultured, miniprepped and performed single/double screening of digests on a 1% agarose gel
Sent 3 IH samples and 1 of both trc-POR (Cam) and lac-CTH1-ycf54-ChlM-trc-POR for sequencing

Made induction plates and induced trc-ChlI1-ChlI2-ChlD-trc-ChlH-GUN4 (Mg-Chelatase) to see if we could get Mg-PPIX formation
Digested and ligated trc-ChlI1-ChlI2-ChlD-ChlH-GUN4 + lac-CTH1-ycf54-ChlM-trc-POR (Streamlined operon)
Bradford assay of trc-POR samples to determine concentration of protein in the supernatant and pellet fractions of the cell lysate

POR is a membrane-associated protein, more (total) dissolved proteins in the supernatant

Extract protochlorophyllide from etiolated seedlings of barley

C₁₈ Sep-Pak used for further purification

Figure 7. Agarose gel (1%) electrophoresis of single (E) and double (EP) digests of trc-POR-ChlP-DVR1-ChlG formed by standard assembly. Both sets of samples showed correct banding for the part and backbone.

Received sequencing results for trc-ChlI1-ChlI2-ChlD-ChlH-GUN4, discovered trc-ChlI1 had not ligated initially but all other genes were present and sequence was confirmed

Stopped streamline operon and digested and ligated trc-ChlI1 to ChlI2-ChlD-ChlH-GUN4

Miniprepped 7 liquid cultures of lac-CTH1-ycf54-ChlM-trc-FNR-fdx (Kan)

Performed single/double digests then ran on 1.5% agarose gel
Sent the 1 successful plasmid off for sequencing
Liquid cultured 8 more colonies of lac-CTH1-ycf54-ChlM-trc-FNR-fdx (Kan)

Performed a functional assay of trc-POR induced cells using protochlorophyllide as substrate

Results were inconclusive and redone by Professor Robert Willows

Received sequencing results for lac-CTH1-ycf54-ChlM-trc-FNR-fdx (Kan) and found frameshift mutation

8 other liquid cultures were miniprepped and screened and 2 successful results were sent off for sequencing

Digestion and ligation of lac-CTH1-ycf54-ChlM-trc-FNR-fdx + trc-POR-ChlP-DVR1-ChlG and backbone swap of trc-POR into Cam
First round of transformations were unsuccessful for lac-CTH1-ycf54-ChlM-trc-FNR-fdx-trc-POR-ChlP-DVR1-ChlG, trc-POR (Cam) and trc-ChlI1-ChlI2-ChlD-ChlH-GUN4; we deduced the issue was with the competent cells we used

Commercial competent cells were used to re-transform samples and succeeded
8 Colonies from each plate were liquid-cultured

Miniprepped and screened through PCR and agarose gel: lac-CTH1-ycf54-ChlM-trc-FNR-fdx-trc-POR-ChlP-DVR1-ChlG, trc-POR (Cam) and trc-ChlI1-ChlI2-ChlD-ChlH-GUN4

Sent 2 samples of trc-ChlI1-ChlI2-ChlD-ChlH-GUN4 and 4 samples of lac-CTH1-ycf54-ChlM-trc-FNR-fdx-trc-POR-ChlP-DVR1-ChlG for sequencing
trc-POR backbone swap did not work so we redigested the original G-Block DNA for backbone insertion into Cam.

Induced trc-ChlI1-ChlI2-ChlD-ChlH-GUN4 (Mg-Chelatase) plasmids into competent cells and used Fluorescence testing to check for enzyme expression and function (formation of Mg-PPIX which is fluorescent).

Growth was too little to yield results at that moment.

We are performing Cas9 purification as part of our collaboration with NTU so we prepared all necessary solutions for Cas9 purification in preparation for the arrival of their biobricks

Figure 8. Agarose gel (1%) electrophoresis of single (E) and double (EP) digests of trc-ChlI1-ChlI2-ChlD-trc-ChlH-GUN4 and trc-POR Cam. trc-ChlI1-ChlI2-ChlD-trc-ChlH-GUN4 showed some successes that will be screened by PCR as the ligation once inserted a 1100bp which is difficult to see on a part this size. trc-POR showed incorrect banding in single digests showing multiple bands.

Figure 9. Agarose gel (1%) electrophoresis of single (E) and double (EP) digests of lac-CTH1-ycf54-ChlM-trc-FNR-fdx-trc-POR-ChlP-DVR1-ChlG standard assembly ligation. The banding size shows correct insertion of trc-POR-ChlP-DVR1-ChlG into lac-CTH1-ycf54-ChlM-trc-FNR-fdx. This forms the second half of our Chlorophyll biosynthesis operon.

Performed the standard assembly of our last 2 parts together for our Chlorophyll plasmid: trc-ChlI1-ChlI2-D-trc-ChlH-GUN4 + lac-CTH1-ycf54-ChlM-trc-FNR-fdx-trc-POR-ChlP-DVR1-ChlG

Screened by colony PCR which was able to narrow down our samples, we miniprepped samples and performed single/double digests for screening

Mg-PPIX induction testing showed high formation of Zn-PPIX which had a similar fluorescence to Mg-PPIX. This dampened out any meaningful results

We proceeded using M9 minimal salt plates for our induction experiments to reduce this problem

Received constructs from NTU and transformed and liquid cultured them, then proceeded to begin with Cas 9 extraction

Figure 10. Agarose gel (1%) electrophoresis of single (E) and double (EP) digests of trc-POR G-block insertion into Cam biobrick backbone for iGEM submission. Samples 4 and 7 showed correct banding of trc-POR biobrick and Cam backbone.

Received sequencing results back for 1st half and last half of complete Chlorophyll pathway plasmid.

Miniprepped sequence confirmed cultures and used PCR to test for presence of genes from each operon of the plasmid (ChlI1, GUN4, trc-FNR-fdx and trc-POR)
Set up induction plates of full chlorophyll plasmid samples

Used SpeedVac to dry out samples in iGEM DNA sample wells for Biobrick sending to iGEM headquarters

Sent trc-ChlH, trc-FNR-fdx, trc-POR, trc-ChlI1-ChlI2-ChlD-trc-ChlH-GUN4, trc-POR-ChlP-DVR1-ChlG and the Complete Chlorophyll pathway biobrick: trc-ChlI1-ChlI2-ChlD-trc-ChlH-GUN4-lac-CTH1-ycf54-ChlM-trc-FNR-fdx-trc-POR-ChlP-DVR1-ChlG
Set up show gel for small parts (trc-ChlH, trc-FNR-fdx and trc-POR)

Transformed lac-CTH1-ycf54-ChlM (w/ and w/out trc-FNR-fdx) into trc-ChlI1-ChlI2-ChlD-trc-ChlH-GUN4 competent cells for functional assay
Set up trc-ChlI1-ChlI2-ChlD-trc-ChlH-GUN4 on M9 minimal salt plates with ALA and IPTG and used fluorescence assay to test for Mg-PPIX

PPIX formation was visible but Mg-PPIX was not

Extracted His-tagged proteins from NTU constructs and ran SDS-PAGE showing successful extraction

Used protein concentrators with a molecular weight cut-off (MWCO) of 100kDa to spin down and remove protein bands under 100 kDA (protein was above that)
Attempted to fuse Cas9 with guide RNA. Unfortunately commercial guide RNA was not functioning to bind to Cas9
Buffer exchange by adding fresh Cas9 working buffer and spinning it down (3 times)

Performed functional assay of Mg-chelatase (trc-ChlI1-ChlI2-ChlD-trc-ChlH-GUN4)

Used large scale inductions; unfortunately cell number was too low for assay

Completed show gel of composite parts
Performed fluorescence assay of our induced Mg-chelatase plasmid incubated with PPIX substrate

Used verified Mg-chelatase subunit proteins (ChlI1, ChlI2, ChlD, ChlH and GUN4) as positive control, showed Mg-PPIX formation
Each subunit was mixed with the induced samples to see if protein expression of any subunit had been a limiting factor in our construct, no individual subunit was shown be limiting factor (none of them showed Mg-PPIX production)

Ran SDS-PAGE gel of our IH3 induced construct

ChlH and ChlI1 expression was visible