Difference between revisions of "Team:Macquarie Australia/Notebook2PSB"

Photosystem II Notebook

This notebook includes the lab-work done to insert the genes to allow E. coli to build a Photosystem-II protein complex.

Project II - Week 1

Restriction sites: Checked all part designs for appropriate prefix/suffix.
Operon 1: Checked operon 1 for features that were present in the design to see how it was broken up (it was broken into three parts so it could actually be synthesised).
Operon 1 part 2: Blastx of protein sequence confirmed correct protein for the gene, however gene sequence did not match that of the operon design. Divergence assumed to be the result of Rob changing the sequence (conserving AA sequence) to assist sequence synthesis. No RBS identified, however alternate RBS have have been used. Yet to be identified.

Project II Week 2

Digested 7 gblocks with EcoRI + PstI:
psbD, psbC, psbA (3 parts of first operon)
psbELJ (operon 2)
psbTB (operon 3)
psbP, psbQR (2 parts of operon 5)
as well as CAM backbone.
Ligated parts into plasmids.
Transformed competent E.Coli cells via heat shocking, and plate out onto Chloramphenicol plates. Incubate.

Checked for update on part 2 of Operon 5, and Operon 4. Check with Rob.W. re: Part 1 of operon 1.

Parts update

Confirmed alternate RBS used in Operon 1 part 2. No further action required.

DNA Oligonucleotide Resuspension and Storage
Upon receiving newly synthesized oligonucleotides, researchers must decide how to resuspend and store the product. Here are some guidelines and recommendations.

Resuspension
Keep in mind most commercially synthesized oligonucleotides are shipped as lyophilized product. Dried DNA is usually very easy to resuspend in an aqueous solution. However, not all oligonucleotides dry identically and some require more time to go into solution than others. It is also possible for the dried oligonucleotide to become dislodged from the tube during shipping. Thus, it very important to spin down every oligonucleotide prior to opening the tube for resuspension.
Aqueous buffer
Resuspend oligos in TE buffer (10mM Tris; 0.1 mM EDTA; pH 8.0) as this buffer will maintain a constant pH. Alternatively, use nuclease-free water. DEPC water will harm oligonucleotides and water from deionizing systems can be overly acidic, with a pH as low as 5.0.
Concentration
Oligonucleotides can be stored at a large range of concentrations. However, concentrations <1 μM may change over time as some of the oligo can adhere to the plastic of the tube. A 5−10 mM solution is generally the highest concentration at which an oligo will go into solution. Resuspension calculations can be made using yield information contained on IDT product specification sheets and on the oligo tube. There you will find the actual yield of the oligonucleotide synthesis in three forms: optical density units (OD); mass (in mg); and copy number (in nmole). At IDT, we routinely resuspend dry oligonucleotides to a storage stock concentration of 100 μM and then dilute a portion of this to create working stock solutions.
To make a 100 μM concentration stock solution: Take the number of nmoles in the tube and multiply that by 10. This will be the number of μL buffer to add to get a 100 μM solution. For example, if you have 9 nmoles oligo, add 90 μL buffer to make a 100 μM solution. If you prefer to work in other units or to resuspend to a different concentration, a Dilution Calculator is available in the SciTools section of the IDT website.
Resuspension
For hard-to-suspend oligos, heat the oligonucleotide at 55°C for 1−5 minutes, then vortex thoroughly. If there is still a visible precipitate in the tube, the sample may contain silica which is a by-product of oligo synthesis. It will not affect the performance of the product, and may be removed through filtration or decanting the supernatant.

Long-term storage

If you would like to use a portion of the oligonucleotide immediately and store the remainder for future use, it is best to resuspend the entire product in Tris-EDTA (TE) buffer, pH 8.0 at the desired stock solution concentration. Take a sufficient volume for immediate use from the stock and dilute it to a working stock concentration. Divide the remaining stock solution into several small aliquots and store at –20°C. Short-term storage
Oligonucleotides that have been resuspended in TE buffer, pH 8.0 can be stored at 4°C for up to 6 months.

Project II - Week 3 Update

Last Week’s results

Successful colonies were observed on Chloramphenicol media for psb-A, psb-C, psb-D, and psb-QR.
No colonies were observed for psb-TB.

Plasmid preparation re-done for psb-TB. To improve transformant yield, the following changes were made to the procedure:
Incubation on ice increased from 10 minutes to 30 minutes The ligation was performed at 37oC instead of 16oC Fragment to vector ratio was increased, using 4:1 instead of 3:1
A plasmid miniprep was performed in triplicate for the successfully transformed samples.
DNA concentration from the miniprep was assessed using a nanodrop.
Samples were digested using EcoRI and PstI.
Gel electrophoresis performed with the 5 extracted samples, non digested samples, and the new and old prep of the psb-TB: hard to read gel, as too much DNA was used. Will redo next week with less DNA, and single/double digests. <-- image goes here -->

Project II - Week 4 Update

Digestion of newly received G-blocks (psb-P, psb-O, psb-MZH, psb-WK) with EcoRI and PstI
Ligation of digested G-blocks into Chloramphenicol backbone, and transformation into E. coli
Re-did gel electrophoresis of last week’s plasmid extracts, with a lower DNA concentration and single/double digests
Today’s results

<-- image goes here -->
Gel electrophoresis shows expected banding for psbC 2 (expected 1456bp), psbQR colony 1 (expected 1092bp), psbELJ colony 2 (expected 756bp). Will send these for sequencing.

Project II - Week 5 Update

Last Week’s results

Of the 4 transformed G-blocks, only Psb-MZH grew colonies on Cam plates, with no colonies observed for psbP, psbO, and PsbWK
Sequencing data showed expected sequence for psbELJ!
Incomplete sequence of psbQR - very short sequences returned (~600bp) for some reason. Have sent for further sequencing with more internal BBF and BBR primers.

Done today

Screening of Psb-A, Psb-D, and Psb-MZH colonies in triplicate, digested with EcoRI and EcoRI/PstI
Probable cause as to why no colonies were observed on Psb-P, Psb-O,and Psb-WK was attributed to insufficient recovery time for competent cells after heat shock
Due to slow/few colony growth for Psb-MZH transformants, combined with the shortened recovery time from 2 hours to 40 minutes due to time constraints
Transformation of Psb-P, Psb-O and Psb-WK was repeated
psbA(x2) and psbMZH(x3) sent for sequencing

Results:

psbA 1 & 2 (expected 1257bp), psbMZH 1, 2, 3 (expected 541bp, faint bands) appear as expected, will send these for sequencing.

Project II - Week 6 Update

Last week’s results

Colonies observed for psb-WK, psb-P, and one colony of psb-TB
Sequencing data for psb-A and psb-MZH shows expected sequence!
Done over the week
psb-ELJ colonies were cultured in induction media

Done today
Ligation/transformation of psb-TB, PsbO and PsbC redone - into Cam backbone
psb-TB redone in case sequencing of last week’s psb-TB colony does not reveal successful incorporation of the biobrick
psb-C redone as digestion of previous psb-C colonies did not show successful incorporation of the biobrick
psb-O redone as no colonies have been isolated for this G-block

Miniprep of colonies containing psb-TB (x1), psb-P (x3), psb-D (x5) and psb-WK (x3)
Nanodrop of each eluted sample to assess DNA concentration
Gel electrophoresis in 1% agarose gel, using 10ng of DNA sample
Gel of both E and EP digestion
SDS-PAGE ran for psb-ELJ cultures
Identified that the incorrect concentrations of Cam backbone to inserts used - possible reason for no/few colonies using psb-TB and psb-O
7.75ng/uL DNA solution, not 10.0ng/uL solution used in previous calculations
6 samples sent for sequencing - 3 x psbP, 3x psbWK

Results:

<-- image goes here -->
SDS-PAGE shows ladder, 5 lanes of induced sample with psbELJ plasmid, then 2 lanes of uninduced control. Target protein sizes are psbE=9.3kDa, psbL=4.4kDa, psbJ=4.2kDa. No protein expression is visible at these sizes, compared to uninduced control.