From 2009.igem.org

Primers were designed to convert MelA into a biobrick. Primers A and F are for either end (including prefix and suffix - highlighted in green). The other primers surround the unwanted restiction sites (highlighted in yellow) with the changed base in red.

These correspond to the parts on the gene shown below. The stars correspond to the two unwanted restriction sites.

Orange Pigment

Used plasmid editor to examine the genes used for the production of orange pigments. The (supposedly) orange-producing pigment from the biobricks:

We also tried a preliminary design for a biobrick we could construct, in case the orange biobrick does not function. This uses the same genetic synthetic pathway for gene consruction:

Transformed Pigments

The MelA plate left on the bench overnight produced a brown-coloured pigment! Growth on media containing IPTG should produce this pigment a lot faster.

Amplification system

Possible plan

We would want to tag the activator proteins then quantify their degradation in some way

GFP fusion: one idea was to create a functional activator-GFP fusion, to which we would then add a C-terminal degradation tag. Thus we could express the activator-GFP-tag in E. coli, and could measure flourescence to quantify activator degradation directly. However, this idea depends on us constructing a functional activator-GFP fusion.
Western blotting: a second idea is to add an N-terminal flag tag to the activator proteins, and then add the C-terminal degradation tag. We could measure the amount of activator using Western blotting and an anti-flag antibody by taking samples over time. However, Western blotting isn't that easy.
Finally, what we're really doing is solving a problem found in the Cambridge 2007 team's system. If we can show that overnight bacterial growth is less hindered with our tagged activators, then we are effectively showing that because of the degradation tags, the activators are indeed being degraded.

We could also attempt to see what this effect has on amplification

Modelling potential - activator half life, change in Pops out?
First we need to dig up the 2007 work and recreate their data, this time with a plate reader that can take OD and fluorescence readings simultaneously.

Activators

Found parts submitted by Cambridge '07 team amplifier project. Three translational units (ribosome binding sites and protein coding sequence) for the three activators. Sequence below, 5' to 3':

Ogr activator from P2 phage: Part I746350

>BBa_I746350 Part-only sequence (237 bp) aaagaggagaaatactagatgtttcattgtcctttatgccagcatgccgcacatgcgcgtacaagtcgctatatcactgacacgacaaaagagcgttatc atcagtgccagaacgtgaattgcagcgccacgttcatcacttatgagtcggtacagcgatacatcgtgaagccgggagaagtccacgccgtaaggccgca cccgttgccatcagggcagcaaattatgtggatgtaa

pag activator from PSP3 phage: Part I746351

>BBa_I746351 Part-only sequence (237 bp) aaagaggagaaatactagatgatgcactgcccgttatgccaaaacgctgcacatgctcgcactagccggtaccttagcaccgaaacgaaagaacgttatc accagtgccaaaacataaattgcggatgtacatttatcacttttgagacactatcaagattcattgtgaaaccggggactgttgatcctgctccgcccca ccccatcagaaaccaacaacagcaactttggctttga

delta activator from phiR73 phage: Part I746352

>BBa_I746352 Part-only sequence (264 bp) aaagaggagaaatactagatgatgcgctgccctttctgtcgtcattcagcgcatacccgcaccagccggtatgtgagtgacaatgtcaaagaaagttatc tccagtgccagaatatttactgttcggcgacatttaaaacgcatgagtcaatttgtgccgtgattcgttctccggtcacggaggaaaaaccagcaccggc aagcacagcaccggctgttgtccgaaaagttaaaggctgttacagctcaccattcaaccattaa

(delete sequence if you think its unnecessary and just leave part names)

Promoters

Available as individual parts, with a GFP reporter or as part of a composite with the activator upstream. Five promoters as used by Cambridge '07:

PF promoter, part: I746360 PO promoter, part: I746361 PP promoter, part: I746362 Psid promoter, part: I746364 PLL promoter, part: I746365

Degradation Tags

three C-terminal tags are well described in Andersen, J. B., Sternberg, C., Poulsen, L. K., Bjorn, S. P., Givskov, M. & Molin, S. (1998). New unstable variants of green fluorescent protein for studies of transient gene expression in bacteria. Appl Environ Microbiol 64, 2240–2246.[http://aem.asm.org/cgi/reprint/64/6/2240], which detailed tagged-GFP degredation.
the tags as described in the paper that we could use are as follows (all DNA sequences are 5' to 3')

LAA: amino acid sequence AANDENYALAA, DNA sequence GCAGCAAACGACGAAAACTACGCTTTAGCAGCT

(primer – CGTCGTTTGCTGCTTTTGATGCGAAATCGTCGA) (currently the "planning" biobrick BBa_M0042)

AAV: amino acid sequence AANDENYAAAV, DNA sequence GCAGCAAACGACGAAAACTACGCTGCAGCAGTT

(primer – CGTCGTTTGCTGCTTTTGATGCGACGTCGTCAA, currently the "planning" biobrick BBa_M0044 with DNA sequence GCAGCAAACGACGAAAACTACGCTGCTGCTGTT)

NOTE: we need to use the biobrick DNA sequence as the DNA sequence described in the paper has a forbidden PstI site

ASV: amino acid sequence AANDENYAASV, DNA sequence GCAGCAAACGACGAAAACTACGCTGCATCAGTT

(primer – CGTCGTTTGCTGCTTTTGATGCGACGTAGTCAA, currently the "planning" biobrick BBa_M0046 with DNA sequence GCTGCAAACGACGAAAACTACGCTGCATCAGTT)

Wet Work

made up 4 15ug/mL tetracycline agar plates
electroporated the following plasmids into our TOP10 competent E. coli cells

K118005
K118006
K118013
K119014
orange plasmid from Dupont strain

plated each biobrick out onto ampicillin plates, plated the orange plasmid onto a tetracycline plate, left to incubate overnight
innoculated 6 single colonies from each of the following cultures, grown over the weekend on plates, in the appropriate antibiotic LB to shake over night in preparation for minipreps and colony PCRs tomorrow

violacein plasmid
melanin plasmid
K152005
R0011

Tuesday

Long Term Aims

1) PIGMENTS

create biobricks

2) AMPLIFICATION

find biobricks
organise system
transform
test
attach to pigments (if time)

3) DEGRADATION TAGS

synthesise
test
attach

4)COLOUR LOGIC

get colour biobricks
attach inducible promoters
test

Team:Cambridge/Notebook/Week3

From 2009.igem.org

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Week 3 - Development

Monday

Primer Design