Team:St Andrews/project/laboratory

Laboratory

Contents 1 Introduction 2 Overview 2.1 The bistable switch 2.2 The CAI-1 sender 3 Week by week progress 3.1 Week 28th June 3.2 Week 5th July 3.3 Week 12th July 3.4 Week 19th July 3.5 Week 23rd July 3.6 Week 30th July 3.7 Week 2nd August 3.8 Week 9th August 3.9 Week 16th August 3.10 Week 23rd August 3.11 Week 30th August 3.12 Week 6th September 3.13 Week 13th September 4 Protocols 4.1 Cell Culture 4.2 Transformation 4.3 Restriction Digest 4.4 Ligation 4.5 PCR 4.6 DNA Extraction 4.7 Annealing of Complementary Primers

Introduction

The synthetic biology portion of our project has provided the undergraduate members of our team with a rare opportunity to work in the lab for an extended period of time. Many of us have gained important skills such as learning to use our time efficiently, planning the use of limited materials and organising experiments effectively. Due to these learning curves the project really kicked off about halfway through our allocated 10 weeks and has been gathering speed as our deadlines approach.

Overview

The lab work has focused on constructing two main biobricks - the bistable switch and the CAI-1 sender.

The bistable switch

The CAI-1 sender

This biobrick is essential to our project. CAI-1, the cholera-specific auto inducing molecule, can switch off the production of cholera toxin by V. cholerae when it is present in high concentrations. To make this biobrick the gene responsible for making the CAI-1 synthase (cqsa) was optimised for E. coli and then synthesised by geneart.

Week by week progress

Week 28th June

Chose roles within the team

Initial lab preparations including making agar, LB broth, antibiotic stock solutions, copied out protocols.

We decided to run test transformations on parts that we would not be using (Part BBa_K09899), as a transformation failure for a critical part would be a problem

First transformation was carried on the 30th of June. This first step was to set the tone for the rest of the project. It failed. Part BBa_K09899. A possible cause of this may be the fact that there was no LB in the agar, this was due to a recent change away from ready mixed LB agar powder (read: we forgot to put nutrients in our media – facepalm).

Week 5th July

Transformed BBa_C0077 resulting in another failure, although this time we made up LB agar properly.

Possible cause – competent cells being used were over three years old. We decided to streak out some of the 2007 competent cells, along with glycerol stocks of regular DH5-alpha and a tube of competent cells that had been kept in the -20 freezer. The old competent cells turned out to be still alive and grew just as vigorously as the regular DH5-alpha and predictably the ones from the -20 were dead. We decided to make up our own competent cells while trying to transform more unuseful parts

Appeared to have successful transformation on the 8th of July. This was a transform of J01003 using the 2007 competent cells. Miniprep and nanodrop readings confirmed presence of plasmid DNA (47.5ng/ul).

We were trained on the safe use of ethidium bromide and the techniques of gel electrophoresis, in addition to the real blast from the past that was figuring out the imaging system software which was made in 1993, on similarly aged hardware.

Following an analytical scale restriction digest of our plasmid DNA using EcoRI and PstI, gel electrophoresis showed plasmid and insert bands for each different amount of DNA we used in the digest. Success!

Week 12th July

We began trying to transform parts that would be required for the bi-stable switch. Dave had a bad feeling about this.

These parts were BBa_F1610, BBa_C0062 and BBa_R0062

Transformation appeared to be successful (colonies observed) but this later proved to be incorrect.

After setting up an overnight culture in antibiotic broth, we miniprepped each part to obtain plasmid DNA. Nanodrop readings varied wildly (this was later discovered to be because the Nanodrop is a difficult beast. It requires scrubbing thoroughly with ethanol and reinitialising before every reading for it to even think of giving you an accurate result, Otherwise the DNA concentration will appear to drop by around 20% with each reading taken)

A restriction digest with EcoRI and PstI followed by gel electrophoresis showed…. “bugger all DNA”. This phrase became commonplace in the following weeks.

Week 19th July

In this week it was discovered that we had been growing mutants, the transformation was not successful and our “competent” cells were in fact, not at all competent. We then found that the calcium chloride concentration used to make our competent cells was incorrect due to a calculation error (biologists can’t handle moles!) so we decided to make up the solution again and make more competent cells. After growing our culture overnight, we diluted 500ul into 50ml LB broth and grew for 2 hours. The protocol suggested measuring the absorbance with the Nanodrop to determine whether or not the cells were in the exponential phase of growth, which we did.

The problem was, the nanodrop gave us a concentration tenfold lower than what we expected. On the second reading, we saw a negative absorbance reading. This was strange. We thought perhaps the cells were dying, but then the ever cynical Dave actually read the Nanodrop manual (unprecedented in this department I think) and found that the protocol gave absorbance values for a 10mm pathlength (the Nanodrop measures a 1mm path).

Learned how to use the SORVALL RC6 Plus SS34 centrifuge (the washing machine sized monster upstairs). Made more competent cells.

Week 23rd July

Made new TBE buffer because an unnamed member of the team disposed of the last lot.

We began having success using competent cells made with the addition of beta mercapto ethanol. This success was short lived, it also turns out that beta mercapto ethanol is toxic, highly pungent and must be diluted to a stock solution in a fume hood.

Week 30th July

Began experimenting with isopropanol miniprep extraction procedure.

Week 2nd August

tansformed B0015, E0240, R0062, C0062, B0032 and R0063.

we compared DNA extraction using isopropanol to extraction using quiagen columns and determined that the isopropanol extracts much more DNA but it as not all plasmid DNA.

A gel was run which showed plasmid but no part.

Began using NEB high efficiency turbo K12 competent E. coli. In general these made life much easier because they allow paarts to be transformed and miniprepped on the same day. They were also much more competent than the cells that we were using before.

Week 9th August

carried out a restriction digest on the results from the isopropanol extraction of E0240, B0032, C0062, R0063, R0062. We aimed for between 0.2 and 1.5 micrograms of DNA in each reaction according to Promega recommendations.

Example recipe:
Deionised water: 15 µl
Restriction Enzyme 10X buffer: 2µl
DNA, tenfold dilution: 2µl
Enzyme - EcoR1: 0.5µl
Enzyme - Pst1: 0.5µl

Transformed C0061 into new turbo cells, also transformed PUC19 control plasmid.

Ran a 1.5% agarose gel of the restriction digests. Even though the isopropanol extracted DNA gave very high nanodrop readings, the bands on the gel weren't any brighter than usual. Insert bands of the correct size were also not observable. We concluded that the nanodrop could become wildly inaccurate at high DNA concentrations, and the isopropanol extraction technique increased the amount of genomic DNA contamination. Decided it was best to verify insert size using PCR with VF2 and VR primers.

Ran another gel, this time with regular miniprep products. Observed some faint bands at the correct insert sizes, but not for all parts. Found that the registry quality control gels also had very faint bands for small parts. Decided to trust the registry and go ahead with assembly.

Colonies from C0061 transformation observed in ring shape, but did not extend to the edge (so unrelated to antibiotic plating) Attributed to cell plating technique. Decided to modify our spreading style, making the "star" in the centre first. Single colonies used to innoculate three 10ml tubes of broth.

Performed miniprep of C0061 transformed previously. "Gunk" observed in culture tubes. Possibly biofilm associated with overgrowth of our Turbo E. coli. Ran restriction digests and gel on this part. Perfectly sized, bright insert bands observed for two of the three cultures taken.

This was the week of the Open golf tournament so the university was shut down for part of the week while the golfers and their fans brought the town to a standstill.

Week 16th August

Began biobrick assembly using NEB assembly kit (3A method)

Upstream Part Digestion
DI Water: 32.5 µl
NEBuffer: 2: 5 µl
BSA: 0.5 µl
DNA: 10 µl
EcoRI: 1 µl
SpeI: 1 µl

Downstream Part Digestion
DI Water: 32.0 µl
NEBuffer: 2: 5 µl
BSA: 0.5 µl
DNA: 10.5 µl
XbaI: 1 µl
PstI: 1 µl

Destination Plasmid Digestion
DI Water: 22.5 µl
NEBuffer: 2: 5 µl
BSA: 0.5 µl
DNA: 20 µl

EcoRI: 1 µl
PstI: 1 µl

Placed at 37C for 10 minutes, then heat inactivated at 80C for 20 minutes.

Ligation Mixture

Distilled Water: 11 µl
T4 Ligase Buffer: 2 µl
Upstream Part Digestion: 2µl
Downstream Part Digestion: 2 µl
Destination Plasmid Digestion:2 µl
T4 DNA Ligase: 1 µl

Placed at room temperature for 15 minutes, then heat inactivated at 80C for 20 minutes. Transformed 5 µl of the ligation into Turbo E. coli.

Primers; f,r,VR and VF2 arrived on the 17th of August allowing PCR based assembly to begin. The primers were all resuspended to make stock solutions.

On the 18th PCR amplification was carried out with VF2 and VR primers on B0032.

The PCR recipe used was

Distilled Water: 25 µl
10X standard taq buffer: 2.5 µl
10mM dNTPs: 0.5 µl
10µM forward primer:0.5 µl
10µM reverse primer:0.5 µl
Template DNA:variable, approximately 3 µl

A miniprep was carried on the O/N cultures from the 16/8/10 assembly.

On the 19th 10mM stock solutions of dNTP stock solutions were made.

PCR amplification of R0063, R0062 and B0015 was carried out. the resulting DNA was of very low concentration.

We ran a gel on digests of the biobrick composites plus some individual parts, the gel was not positive.

We received a stern warning from Olivia that we should be more complete when completing our lab notebooks, a mental note of this information was made.

We digested B0015 from the PCR product. We also digested R0063, R0062, B0015, C0062, C0061, E00240 and confirmed our results on a gel. There was a cock up with the promoter digestions so they were repeated. We then began ligating the different parts of our bistable switch together.

Week 23rd August

Transformed our ligations, made an overnight, colony PCR (Drew Endy protocol)

colony PCR reaction mix:
water 6.6 µl
Taq buffer 1 µl
10x dNTP 1 µl
template 1 µl
fwd primer 0.25 µl
rev primer 0.25 µl

PCR purification, gel - negative results :(

We have not been getting the DNA yields that we would like using the column miniprep technique so this week we experimented with ethanol precipitation using the following protocol

once you have done the steps adding P1 P2 and N3 buffers from the standard miniprep protocol, and you have spun it for 10mins at 12,000 take that supernatant and add it to new eppendorf tubes.

add 650ul isopropanol, then put them in the freezer for a few minutes

spin for 10min at 12,000

there should be a tiny tiny faint white pellet - it is often really hard to see, or not there at all, it can be a bit hit and miss.

on the 27th we did colony PCR of CO261 and I13401.
We also did digests of e0240 and i13401. remove the supernatant carefully

add 300ul 70% ethanol

resuspend the pellet

spin for 5 min at 12,000

remove the supernatant, and leave the eppendorf lids open on the bench for 5 to 10 mins to allow the ethanol to evaporate off. resuspend in 40ul of distilled water/ 10mM Tris or TE.

This protocol led to massively increased DNA yield however it appeared that the majority of it was not plasmid.

Week 30th August

we inoculated LB using a c0261 colony and allowed it to grow. we followed this with a miniprep.

this week our cqsa gene arrived from MrGene. we transformed some cells with it and then did a miniprep.we then digested the miniprep using the standard NEB protocol. this was followed by ligating the cqsa into the standard biobrick plasmid.
we also did some colony pcr and ran more gels.

Week 6th September

Week 13th September

Protocols

We used a number of protocols from OpenWetWare in addition to the manufacturer's recommended protocols for our materials.

Cell Culture

OpenWetWare [http://openwetware.org/wiki/Bacterial_cell_culture Bacterial Cell Culture]

Transformation

NEB Turbo Competent E. coli

Clontech Stellar Competent E. coli.

Restriction Digest

Promega Enzymes

NEB Biobrick Assembly Kit (Gingko Bioworks)

Ligation

NEB Biobrick Assembly Kit (Gingko Bioworks)

PCR

OpenWetWare [http://openwetware.org/wiki/Knight:Colony_PCR Knight - Colony PCR]

NEB Taq

Finnzymes Phusion

DNA Extraction

Quiagen Miniprep

Annealing of Complementary Primers

OpenWetWare [http://openwetware.org/wiki/Endy:Annealing_complementary_primers Endy - Annealing Complementary Primers]