Ministers and MPs are heading to Uxbridge and South Ruislip, where Mr Johnson quit as MP, to try to stop Labour seizing it by over-turning a Tory majority of more than 7,000.
They are already highlighting the controversial extension of the Mayor’s ultra low emission zone to outer London boroughs.
Tory Party chairman Greg Hands, campaigning in the constituency, argued that the ulez expansion was “very, very unpopular around here”.
He added: “This is a great opportunity to send a message to Mayor Khan that Ulez is not welcome here.”
Another Tory source said: “This will be a referendum on Sadiq Khan’s Ulez.”
Senior Conservatives believe that if they can hold onto Uxbridge and South Ruislip, it would pile pressure on Sir Keir Starmer to intervene to force Mr Khan to scale back his expansion of Ulez due to come into force at the end of August.
Several town halls are seeking to block the extension through court action.
Shadow health secretary Wes Streeting, MP for Ilford North, speaking on LBC Radio, said he backed the Mayor’s move to tackle toxic air in the capital and welcomed a “more generous scrappage scheme” announced for motorists forced to change their vehicle due to the wider Ulez zone.
The Government was expected to move writs on Wednesday, or possibly later this week, to trigger by-elections in July in the west London seat and in the North Yorkshire constituency of Selby and Ainsty after one of Mr Johnson’s close allies Nigel Adams MP also resigned.
The Conservatives poured activitists into the Mid Bedfordshire constituency over the weekend, where they are defending a 24,664 majority, after the sitting MP Nadine Dorries also announced that she was quitting.
But as of early Wednesday morning, she had yet to formally resign, sparking speculation that she may stay on to cause more chaos for Rishi Sunak.
A long delay would mean that the Prime Minister may not be able to hold all three by-elections on the same day, which might have limited the political fall-out if the Tories lose two or three of them.
Conservative peer and pollster Lord Robert Hayward told The Standard: ““All three of these by-elections are in play.
“All parties will treat them as very serious contests.”
He added: “In a difficult set of circumstances, he (the PM) has been trying to show that he is in charge of the Government and will therefore want the best possible results in each of the constituencies.
“There is no question that these are important for Keir as well because he has to be able to show that the Labour Party is making progress across the country.”
Lord Hayward stressed that in the short-run, Mr Sunak is faced with a “difficult combination of events” after the shock resignation of Mr Johnson on Friday.
But he believes that the ex-PM’s departure, and of his allies, may in the long-run turn into Mr Sunak’s “advantage”, partly as it weakens his support in Parliament.
Holding onto Uxbridge and South Ruislip, where the Tory majority in 2019 was 7,210, could prove more challenging than in Selby and Ainsty, where the majority was 20,137.
But the Conservatives have a history of by-election successes in the west London seat, with Mr Johnson’s predecessor John Randall, now a peer, winning there in 1997 shortly after Tony Blair’s landslide victory.
His predecessor Sir Michael Shersby was also elected at a by-election in 1972.
Demographic changes in the constituency, though, may make it harder for the Tories to win this time.
The Tory majority in Mid-Beds was 24,664 in 2019.
However, the Liberal Democrats, who are targeting the seat, won the Tiverton and Honiton by-election last June, overturning a Conservative majority of more than 24,000
Mr Johnson formally quit on Monday under the ancient tradition of taking the Chiltern Three Hundreds.
“The Chancellor of the Exchequer has this day appointed Alexander Boris de Pfeffel Johnson to be Steward and Bailiff of the Three Hundreds of Chiltern,” the Treasury announced.
Mr Adams was made Steward and Bailiff of the Manor of Northstead.
The “Steward and Bailiffs of the Chiltern Hundreds and of the Manor of Northstead” were positions traditionally paid for by the Crown.
In modern times they are unpaid, formal titles that are applied for when an MP needs to disqualify themselves from the Commons.
An elected MP has no right to resign, explained Commons officials, so unless they die or are expelled they must become disqualified if they wish to retire before the end of a Parliament.
By law, taking on one of these titles immediately bars a person from being an MP.